Multivalent heteromultimer scaffold design and constructs

ABSTRACT

Provided herein are multifunctional heteromer proteins. In specific embodiments is a heteromultimer that comprises: at least two monomeric proteins, wherein each monomeric protein comprises at least one cargo polypeptide, attached to a transporter polypeptide, such that said monomeric proteins associate to form the heteromultimer. These therapeutically novel molecules comprise monomers that function as scaffolds for the conjugation or fusion of therapeutic molecular entities resulting in the creation of bispecific or multivalent molecular species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/411,353, filed Mar. 2, 2012, now U.S. Pat. No. 9,499,605, whichclaims the benefit of U.S. Provisional Patent Application No.61/449,016, filed Mar. 3, 2011, which are incorporated herein byreference in their entireties.

FIELD OF INVENTION

The field of the invention is the rational design of a scaffold forcustom development of biotherapeutics.

DESCRIPTION OF RELATED ART

In the realm of therapeutic proteins, antibodies with their multivalenttarget binding features are excellent scaffolds for the design of drugcandidates. Advancing these features further, designed bispecificantibodies and other fused multispecific therapeutics exhibit dual ormultiple target specificities and an opportunity to create drugs withnovel modes of action. The development of such multivalent andmultispecific therapeutic proteins with favorable pharmacokinetics andfunctional activity has been a challenge.

Human serum albumin (HSA, or HA), a protein of 585 amino acids in itsmature form is responsible for a significant proportion of the osmoticpressure of serum and also functions as a carrier of endogenous andexogenous ligands. The role of albumin as a carrier molecule and itsstable nature are desirable properties for use as a carrier andtransporter of polypeptides in vivo.

Human serum albumin possesses many desirable characteristics. HSA isfound throughout the body, but more specifically in the interstitialspace and in blood at serum concentrations of 40 g/L which is equivalentto 0.7 mM (Yeh et al., Proc. Natl. Acad. Sci. USA, 89:1904-1908 (1992)).HSA is considered to be the most abundant protein of the serum and isresponsible for maintaining osmolarity. HSA has favorablepharmacokinetic properties and is cleared very slowly by the liver andkidney displaying in vivo half-lives up to several weeks (Yeh et al.,Proc. Natl. Acad. Sci. USA, 89:1904-1908 (1992); Waldmann, T. A.,Albumin Structure, Function and Uses, pp. 255-273 (1977); Sarav et al.,J Am Soc Nephrol 20:1941-1952(2009)). HSA lacks enzymatic activity andantigenicity thereby eliminating potentially undesirable side effects.HSA acts as a carrier for endogenous as well as exogenous ligands.Combined, these features can be extended, at least partially, ontoalbumin based fusion protein. The poor pharmacokinetic propertiesdisplayed by therapeutic proteins can then be circumvented.

SUMMARY OF THE INVENTION

Provided herein are multifunctional heteromultimers and methods todesign them. In certain embodiments are heteromultimers, eachheteromultimer comprising: at least a first monomer unit that comprisesat least one cargo molecule, and a first transporter polypeptide; and atleast a second monomer unit that comprises at least one cargo moleculeand a second transporter polypeptide; wherein at least one transporterpolypeptide is derived from a monomeric protein and wherein saidtransporter polypeptides self-assemble to form a quasi-native structureof said monomeric protein or analog thereof. In certain embodiments, atleast one cargo molecule is a drug, or a therapeutic agent. In certainembodiments, at least one cargo molecule is a biomolecule. In anembodiment, the at least one biomolecule is a DNA, RNA, PNA orpolypeptide. In an embodiment, at least one cargo molecule is apolypeptide. In certain embodiments, each monomeric transporterpolypeptide is unstable and preferentially forms a heteromultimer withat least one other transporter polypeptide. In certain embodiments, eachmonomeric transporter polypeptide is stable and preferentially forms aheteromultimer with at least one other transporter polypeptide. Incertain embodiments, the heteromultimerization interface comprises atleast one disulfide bond. In certain embodiments, theheteromultimerization interface does not comprise a disulfide bond.

In specific embodiments is a heteromultimer that comprises: at least twomonomers, wherein each monomer comprises at least one cargo moleculeattached to a transporter polypeptide, such that said monomersself-assemble to form the heteromultimer. In certain embodiments is aheteromultimer that comprises: at least two monomeric proteins, whereineach monomeric protein comprises at least one cargo polypeptide,attached to a transporter polypeptide, wherein at least one transporterpolypeptide is derived from a monomeric protein and wherein saidtransporter polypeptides self-assemble to form a quasi-native structureof said monomeric protein or analog thereof. In certain embodiments is aheteromultimer that comprises: at least two monomeric proteins, whereineach monomeric protein comprises at least one cargo polypeptide attachedto a transporter polypeptide, such that said monomeric proteinsself-assemble via the transporter polypeptide to form theheteromultimer, and wherein at least one transporter polypeptide isderived from a monomeric protein and wherein said transporterpolypeptides self-assemble to form a quasi-native structure of saidmonomeric protein or analog thereof. In certain embodiments, theheteromultimer is a heterodimer. In an embodiment, the heteromultimer isbispecific. In an embodiment, the heteromultimer is multispecific. Incertain embodiments, the heteromultimer is bivalent. In an embodimentthe heteromultimer is multivalent. In an embodiment, the heteromultimeris multifunctional. In certain embodiments, at least one transporterpolypeptide is not derived from an antibody. In certain embodiments, thetransporter polypeptides are not derived from an antibody. In certainembodiments, the transporter polypeptides are derivatives of albumin. Incertain embodiments of the heteromultimer described herein, thetransporter polypeptides are derived from human serum albumin (HSA orHA) of SEQ ID No. 1. In certain embodiments of the heteromultimerdescribed herein, the transporter polypeptides are derived fromalloalbumins (HAA). In certain embodiments of the heteromultimerdescribed herein, the transporter polypeptides are derived from sequencehomologous to the human serum albumin (HSA or HA) of SEQ ID No. 1.

In some embodiments of the heteromultimer described herein, thetransporter polypeptides are derivatives of an annexin protein. In anembodiment, the transporter polypeptides are derived from differentannexin proteins. In certain embodiments, the transporter polypeptidesare derived from the same annexin protein. In an embodiment, at leastone transporter polypeptide is derived from Annexin A1 or lipocortin I.In certain embodiments of the heteromultimer, all transporterpolypeptides are derived from Annexin A1 of SEQ ID NO: 14. In certainembodiments of the heteromultimer, at least one transporter polypeptidesis derived from a sequence homologous to SEQ ID NO: 14. In anembodiment, at least one transporter polypeptide is derived from AnnexinA2 or annexin II. In certain embodiments of the heteromultimer, alltransporter polypeptides are derived from Annexin A2 or lipocortin II.In an embodiment, at least one transporter polypeptide is derived fromAnnexin like protein. In certain embodiments of the heteromultimer, alltransporter polypeptides are derived from Annexin like protein. In anembodiment, at least one transporter polypeptide is derived from thegroup comprising Annexin A1-Annexin A7. In an embodiment of theheteromultimer described herein, all transporter polypeptides arederived from the group comprising Annexin A1-Annexin A7. 14. In certainembodiments, the first annexin based transporter polypeptide has asequence comprising SEQ ID NO:15, and the second annexin basedtransporter polypeptide has a sequence comprising SEQ ID NO: 16.

In some embodiments of the heteromultimer described herein, thetransporter polypeptides are derivatives of transferrin. In anembodiment, at least one transporter polypeptide is derived fromtransferrin. In certain embodiments of the heteromultimer, at least onetransporter polypeptides are derived from transferrin of SEQ ID NO: 19or analog thereof. In certain embodiments of the heteromultimer, atleast one transporter polypeptide is derived from a polypeptide sequencehomologous to the transferrin. In certain embodiments of theheteromultimer described herein, at least one transporter polypeptide isderived from apo-transferrin. In certain embodiments, the firsttransferrin based transporter polypeptide has a sequence comprising SEQID NO:15 and the second transferrin based transporter polypeptide has asequence comprising SEQ ID NO: 16.

In certain embodiments of the heteromultimer, at least one cargomolecule is a cargo polypeptide. In an embodiment of the heteromultimerdescribed herein, all cargo molecules are cargo polypeptides. In certainembodiments, the cargo polypeptides are therapeutic proteins orfragments or variants thereof. In certain embodiments, the cargopolypeptides are antigens or fragments or variants thereof. In certainembodiments, the cargo polypeptides are antigen receptors or fragmentsor variants thereof. In some embodiments, the cargo polypeptide is anantibody, an antibody domain, a ligand or a receptor that binds a targetpolypeptide. In some embodiments, at least one cargo polypeptide isfused to the transporter polypeptide. In certain embodiments, at leastone cargo polypeptide is attached to the N-terminus of the transporterpolypeptide. In some embodiments, at least one cargo polypeptide isattached to the C-terminus of the transporter polypeptide. In someembodiments, at least one cargo polypeptide is chemically linked to thetransporter polypeptide. In some embodiments of the heteromultimersdescribed herein, at least one cargo polypeptide comprises GLP-1 orfragment or variant thereof. In some embodiments, at least one cargopolypeptide comprises glucagon or fragment or variant thereof. In anembodiment, at least one cargo polypeptide comprises an EGF-A likedomain.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide. In certain embodiments, theheteromultimer is a heterodimer. In an embodiment, the heteromultimer ismultispecific. In an embodiment, the heteromultimer is bispecific. Incertain embodiments of the heteromultimer, the transporter polypeptidesare derivatives of the same protein. In certain embodiments, thetransporter polypeptides are derivatives of albumin. In certainembodiments of the heteromultimer described herein, the transporterpolypeptides are derived from human serum albumin of SEQ ID No. 1. Incertain embodiments, the transporter polypeptides are derivatives of anannexin. In an embodiment, the transporter polypeptides are derivativesof Annexin A2. In some embodiments, the transporter polypeptides arederivatives of transferrin.

In certain embodiments, are heteromultimers, each heteromultimercomprising: at least a first monomeric protein that comprises at leastone cargo polypeptide and a first transporter polypeptide comprising afirst segment of human serum albumin; and at least a second monomericprotein that comprises at least one cargo polypeptide, fragment and asecond transporter polypeptide comprising a second segment of humanserum albumin; wherein said transporter polypeptides self-assemble toform a quasi-native structure of albumin or analog thereof. In certainembodiments, the first and second segments of human serum albumin arefrom non-overlapping regions of the protein. In certain embodiments,there is an overlap between the sequences of the first and secondsegments of human serum albumin. In some embodiments, the overlap is a5% overlap. In an embodiment, the overlap is a 10% overlap. In certainembodiments, the first segment of human serum albumin comprises asequence of SEQ ID NO:2, and the second segment of human serum albumincomprises a sequence of SEQ ID NO: 3. In certain embodiments, the firstsegment of human serum albumin comprises a sequence of SEQ ID NO:8, andthe second segment of human serum albumin comprises a sequence of SEQ IDNO: 10.

In certain embodiments, are heteromultimers, each heteromultimercomprising: at least a first monomeric protein that comprises at leastone cargo polypeptide and a first transporter polypeptide comprising asequence of SEQ ID NO:2; and at least a second monomeric protein thatcomprises at least one cargo polypeptide, and a second transporterpolypeptide comprising a sequence of SEQ ID NO: 3. In certainembodiments, are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide comprising a sequence ofSEQ ID NO:8; and at least a second monomeric protein that comprises atleast one cargo polypeptide and a second transporter polypeptidecomprising a sequence of SEQ ID NO: 10. In certain embodiments of theheteromultimer described herein, at least one transporter polypeptide isderived from alloalbumins. In certain embodiments, both transporterpolypeptides are derived from alloalbumins. In certain embodiments, alltransporter polypeptides are derivatives of the same alloalbumin. Insome other embodiments, the transporter polypeptides are derivatives ofdifferent alloalbumins. In some embodiments, each transporterpolypeptide is an alloalbumin derivative based on an alloalbuminselected from Table 2. In certain embodiments, the first monomericprotein comprises two cargo polypeptides. In some embodiments, thesecond monomeric protein comprises two cargo polypeptides. In someembodiment, at least one of the monomeric proteins is engineered byintroducing mutations. In certain embodiments, the introduced mutationsimprove the functionality of the monomeric protein as compared to thenative, non-mutated form of the monomer. In certain embodiments theintroduced mutations improve one or more of the stability, half-life andheteromultimer formation of the transporter polypeptide.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide. In certain embodiments, at least onecargo polypeptide is selected from the proteins listed in Table 2 orfragments, variants or derivatives thereof. In certain embodiments, atleast one cargo polypeptide is selected from ligand, receptor, orantibody to one or more proteins listed in Table 2, or fragment, variantor derivative of said ligand, receptor or antibody. In certainembodiments, at least one cargo polypeptide targets a cell surfaceantigen from the group consisting of CD19, CD20, CD22, CD25, CD30, CD33,CD40, CD56, CD64, CD70, CD74, CD79, CD105, Cd138, CD174, CD205, CD227,CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphB2, EphA2,FAP, integrin, Mesothelin, EGFR, TAG-72, GD2, CAIX, 5T4. In certainembodiments, are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein at least one at least one cargopolypeptide is an antibody, or fragment or variant thereof. In certainembodiments, all cargo polypeptides are antibodies or fragments orvariants thereof. In some embodiments, the cargo polypeptide is anantibody that binds to a protein listed in Table 2. In some embodiments,the antibody fragment comprises antibody Fc or Fab or Fv region. In someembodiment the cargo polypeptide is a non-antibody protein likenanobodies, affibody, maxibody, adnectins, domain antibody, evibody,ankyrin repeat proteins, anticalins, camlids or ligand protein orpolypeptide binding to a therapeutically relevant target. In someembodiments, the antibody or its fragment is derived from animmunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE,and IgM. In certain embodiments, the IgG is of subtype selected fromIgG1, IgG2a, IgG2b, IgG3 and IgG4. In certain embodiments, the antibodyis multispecific.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein at least one cargo polypeptideis a therapeutic antibody. In some embodiments of the heteromultimersdescribed herein, at least one cargo polypeptide is a therapeuticantibody or fragment or variant thereof, wherein the antibody isselected from antibody is selected from abagovomab, adalimumab,alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab,bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol,certuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumabozagamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab,lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab,nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab,panitumumab, pertuzumab, ranizumab, reslizumab, rituximab, teplizumab,toclizumab, tositumomab, trastuzumab, Proxinium, Rencarex, ustekinumab,and zalutumumab. In certain embodiments, the therapeutic antibody bindsa disease related target antigen such as cancer antigen, inflammatorydisease antigen or a metabolic disease antigen. In certain embodiments,the target antigen could be a protein on a cell surface and the cellcould belong to the group of B-cell, T-cell, stromal cell, endothelialcell, vascular cell, myeloid cell, hematopoietic cell or carcinoma cell.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomer that comprises at least one cargo molecule,fragment; and at least a second monomer that comprises at least onecargo molecule and a second transporter polypeptide, wherein at leastone cargo polypeptide is an enzyme, enzyme inhibitor, hormone,therapeutic polypeptide, antigen, radiotoxin and chemotoxin inclusive ofbut not restricted to neurotoxins, interferons, cytokine fusion toxinsand chemokine fusion toxins, cytokine, antibody fusion protein orvariant or fragment thereof. In some embodiments of the heteromultimersdescribed herein, at least one cargo polypeptide comprises GLP-1 orfragment or variant thereof. In some embodiments, at least one cargopolypeptide comprises glucagon or fragment or variant thereof. In anembodiment, at least one cargo polypeptide comprises an EGF-A likedomain. In certain embodiments, the toxin is an immunotoxin such asDenileukin diftitox and Anti-CD22 immunotoxin such as CAT-3888 andCAT-8015. In certain embodiments, the toxin is saporin. In someembodiments, the toxin is a mitotoxin. In some embodiments, the toxin isa diphtheria toxin. In some embodiments, the toxin is botulinux toxintype A. In some embodiments, the toxin is ricin or a fragment there of.In some embodiments, the toxin is a toxin from RTX family of toxins.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein the cargo polypeptide isattached to the transporter polypeptide by chemical conjugation, nativeligation, chemical ligation, a disulfide bond or direct fusion or fusionvia a linker. In certain embodiments, linkers for attaching cargomolecules such as cargo polypeptides to transporter polypeptides areselected from the linkers described in U.S. Pat. Nos. 5,482,858,5,258,498 and U.S. Pat. No. 5,856,456, US2009060721, U.S. Pat. Nos.6,492,123, 4,946,778, 5,869,620, 7,385,032, 5,073,627, 5,108,910,7,977,457, 5,856,456, 7,138,497, 5,837,846, 5,990,275, EP1088888incorporated by reference herein.

Provided herein are host cells comprising nucleic acid encoding aheteromultimer described herein. In certain embodiments, the nucleicacid encoding the first monomeric protein and the nucleic acid encodingthe second monomeric protein are present in a single vector. In certainembodiments, the nucleic acid encoding the first monomeric protein andthe nucleic acid encoding the second monomeric protein are present inseparate vectors.

Provided herein is a method of making a heteromultimer, wherein saidmethod comprises: culturing a host cell described herein such that thenucleic acid encoding a heteromultimer described herein is expressed;and recovering the heteromultimer from the cell culture. In someembodiments, the host cell is a prokaryotic cell or a eukaryotic cell.In some embodiments, the host cell is E. coli. In certain embodiments,the host cell is yeast cell. In some embodiments, the yeast is S.cerevisiae. In some embodiments, the yeast is Pichia. In a certainembodiment, the yeast is Pichia pastoris. In some embodiments, the yeastis glycosylation deficient, and/or protease deficient. In someembodiments, the host cell is a bacterial cell. In some embodiments, thehost cell expressing a heteromultimer descried herein is a mammaliancell. In certain embodiments, the mammalian cell is a CHO cell, a BHKcell, NSO cell, COS cell or a human cell.

Provided is a pharmaceutical composition that comprises a heteromultimerdescribed herein and a pharmaceutically acceptable adjuvant. Alsoprovided are methods of treating an individual suffering from a diseaseor disorder, said method comprising administering to the individual aneffective amount of a formulation or pharmaceutical compositiondescribed herein. In certain embodiments is a method of treating cancerin a patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In some embodiments is a method of treating an immune disorder in apatient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.Also provided is a method of treating an infectious disease in apatient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating a cardiovascular disorderin a patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating a respiratory disorder ina patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating a metabolic disorder in apatient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating one or more of Congenitaladrenal hyperplasia, Gaucher's disease, Hunter syndrome, Krabbe disease,Metachromatic leukodystrophy, Niemann-Pick disease, Phenylketonuria(PKU), Porphyria, Tay-Sachs disease, and Wilson's disease in a patient,said method comprising administering to the patient a therapeuticallyeffective amount of a heteromultimer described herein.

Provided is a kit for detecting the presence of a biomarker of interestin an individual, said kit comprising (a) an amount of a heteromultimerdescribed herein, wherein said heteromultimer comprises at least onecargo polypeptide such that said cargo polypeptide is capable of bindingto the biomarker of interest; and (b) instructions for use.

Provided herein are heteromultimer proteins that comprise at least twomonomeric proteins, wherein each monomeric protein comprises at leastone cargo polypeptide, and an albumin based polypeptide, such that saidmonomeric proteins self-assemble to form the heteromultimer.

In certain embodiments, the cargo polypeptide is fused to the albumin oralloalbumin based transporter polypeptide. In some embodiments, thecargo polypeptide is fused to the transferrin based transporterpolypeptide. In certain embodiments, the cargo polypeptide is fused tothe annexin based transporter polypeptide. In some embodiments, thefusion is at the N terminus of the transporter polypeptide. In certainembodiments, the fusion is at the C terminus of the transporterpolypeptide. In some embodiments, the fusion involves a bridging linkeror spacer molecule. In some embodiments, the cargo polypeptide ischemically conjugated to the transporter polypeptide. In certainembodiments, the cargo polypeptide is attached to the transporterpolypeptide by means of chemical ligation or a disulfide bond.

Provided herein are heteromultimer proteins that comprise at least twomonomeric proteins, wherein each monomeric protein comprises at leastone cargo polypeptide, and a transporter polypeptide, such that saidtransporter polypeptides self-assemble to form the heteromultimer. Insome embodiments, each transporter polypeptide is an alloalbumin basedpolypeptide, such that said alloalbumin based polypeptides self-assembleto form the heteromultimer. In some embodiments, each transporterpolypeptide is a transferrin based polypeptide. In some embodiments,each transporter polypeptide is an annexin based polypeptide. In certainembodiments, each monomeric transporter polypeptide is unstable andpreferentially forms a heteromultimer with at least one othertransporter polypeptide.

In some embodiments, a heteromultimer described herein is a heterodimer.In some embodiments cargo polypeptide is an antibody, enzyme, hormone,therapeutic polypeptide, antigen, chemotoxin, radiotoxin, cytokine orvariant or fragment thereof. In some embodiments, the cargo polypeptideof one monomeric protein functions in synergy with the cargo polypeptideof another monomeric protein.

Provided herein are heteromultimer proteins that comprise at least twomonomeric proteins, wherein each monomeric protein comprises at leastone cargo polypeptide, and an annexin based polypeptide, such that saidannexin based polypeptides self-assemble to form the heteromultimer witha quasi-native structure of annexin or analog thereof. In someembodiments, the annexin is Annexin A1. In some embodiments, aheteromultimer described herein is a heterodimer. In some embodimentscargo polypeptide is an antibody, enzyme, hormone, therapeuticpolypeptide, antigen, chemotoxin, radiotoxin, cytokine, ligand to areceptor, receptor or variant or fragment thereof. In some embodiments,the cargo polypeptide of one monomeric protein functions in synergy withthe cargo polypeptide of another monomeric protein. In some embodimentsthe cargo polypeptide can be an agonist or antagonist to the cargopolypeptide of another monomeric protein.

Provided herein are heterodimer proteins that comprise at least twomonomeric fusion proteins, wherein each monomeric fusion proteinscomprises at least one cargo polypeptide fused to an albumin derivedpolypeptide, such that said albumin derived polypeptides self-assembleto form the multifunctional heterodimer. In certain embodiments areheterodimeric proteins comprising a first monomer which comprises atleast one cargo polypeptide fused to an albumin derived polypeptide; anda second monomer that comprises at least one cargo polypeptide fused toan albumin derived polypeptide. In certain embodiments, the at least onecargo polypeptide of the first monomer is different from the at leastone cargo polypeptide of the second monomer. In certain embodiments, theat least one cargo polypeptide of the first monomer is the same as theat least one cargo polypeptide of the second monomer.

In certain embodiments are heteromultimer proteins that comprise atleast two monomeric fusion proteins, wherein each monomeric fusionproteins comprises at least one cargo polypeptide fused to analloalbumin derived polypeptide, such that said alloalbumin derivedpolypeptides self-assemble to form the multifunctional heteromultimer.In certain embodiments are heteromultimer proteins that comprise atleast two monomeric fusion proteins, wherein each monomeric fusionproteins comprises at least one cargo polypeptide fused to a transferrinderived polypeptide, such that said transferrin derived polypeptidesself-assemble to form the heteromultimer. In certain embodiments areheteromultimer proteins that comprise at least two monomeric fusionproteins, wherein each monomeric fusion proteins comprises at least onecargo polypeptide fused to an annexin derived polypeptide, such thatsaid annexin derived polypeptides self-assemble to form theheteromultimer. In certain embodiments, the annexin is Annexin A2.

In certain embodiments are heteromultimer proteins comprising a firstmonomer which comprises at least one cargo polypeptide fused to analloalbumin derived polypeptide; and a second monomer that comprises atleast one cargo polypeptide fused to an alloalbumin derived polypeptide.In certain embodiments, the at least one cargo polypeptide of the firstmonomer is different from the at least one cargo polypeptide of thesecond monomer. In certain embodiments, the at least one cargopolypeptide of the first monomer is the same as the at least one cargopolypeptide of the second monomer.

Provided herein is a heteromultimer that comprises: at least twomonomers, each comprising a transporter polypeptide and optionally atleast one cargo molecule attached to said transporter polypeptide,wherein each transporter polypeptide is obtained by segmentation of awhole protein such that said transporter polypeptides self-assemble toform quasi-native whole protein. In certain embodiments, theheteromultimer is multispecific. In certain embodiments, the transporterpolypeptides are not derived from an antibody. In some embodiments, eachmonomer preferentially forms the heteromultimer as compared to a monomeror a homomultimer. In an embodiment of the heteromultimer, at least onecargo molecule is a therapeutic agent, or a biomolecule. In someembodiments, at least one cargo molecule is a biomolecule which isselected from a polypeptide, DNA, PNA, or RNA. In some embodiments, eachtransporter polypeptide is a derivate of albumin or alloalbumin. In anembodiment, each transporter polypeptide is a derivate of annexin. Incertain embodiments, each transporter polypeptide is a derivate oftransferrin.

In certain embodiments are pharmaceutical formulations that comprise analbumin-based and/or alloalbumin-based heteromultimeric proteindescribed herein and a pharmaceutically acceptable diluent or carrier.In certain embodiments are pharmaceutical formulations that comprise atransferrin-based heteromultimeric protein described herein and apharmaceutically acceptable diluent or carrier. In certain embodimentsare pharmaceutical formulations that comprise an annexin-basedheteromultimeric protein described herein and a pharmaceuticallyacceptable diluent or carrier. In certain embodiments are pharmaceuticalformulations that comprise an Annexin-A2 based heteromultimeric proteindescribed herein and a pharmaceutically acceptable diluent or carrier.In certain embodiments, a formulation described herein is provided aspart of a kit or container. In certain embodiments, the kit or containeris packaged with instructions pertaining to extended shelf life of thetherapeutic protein. In some embodiments, a heteromultimer describedherein is used in a method of treating (e.g., ameliorating) preventing,or diagnosing a disease or disease symptom in an individual, comprisingthe step of administering said formulation to the individual.

Provided herein is a method of obtaining fusion protein scaffolds with aknown number of conjugation sites based on any transport protein ofinterest.

Also provided are transgenic organisms modified to contain nucleic acidmolecules described herein to encode and express monomeric fusionproteins described herein.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

In drawings which illustrate embodiments of the invention,

FIG. 1 depicts the structure of the Human Serum Albumin (HSA) molecule.The alpha helical sections of the secondary structure are shownschematically along with the bonds represented as sticks.

FIG. 2 is a plot of buried solvent accessible surface area at theinterface of two albumin-based polypeptides.

FIG. 3 depicts two albumin-based polypeptides expressed separately. Thetwo polypeptides are shown in light and dark grey respectively. Eachpolypeptide comprises two fusion sites for functional cargo proteins andthese sites are represented as spheres. The disulphide residues instructure are shown as sticks.

FIG. 4 is a schematic representation of bispecific and othermultifunctional therapeutics based on the multispecific heteromultimerdescribed herein. The albumin-based, or alloalbumin-based polypeptidesare denoted A1 and A2. Multifunctional heteromultimers are obtained byconjugating antigen binding motifs, cytokines and other forms ofsignaling molecules, chemotoxin, radiotoxins or other functionallyrelevant immunoconjugates to N and/or C terminal sites on A1 and A2 andthis is represented by the + symbol.

FIG. 5 is a schematic of a bispecific antibody derived from aheterodimeric Fc domain. Albumin or alloalbumin based polypeptides areconnected to the C-terminal of the Fc to selectively drive the formationof heterodimers.

FIGS. 6A-6C show native gel electrophoresis profiles of full-length HSAand heterodimer scaffolds Albumin-based heteromultimer-1 (ABH1) andAlbumin-based heteromultimer-2 (ABH2) formed by coexpression of HSAbased transporter polypeptides.

FIG. 7 shows stability of wild type HSA and heterodimer scaffolds ABH1and ABH2 studied using Differential Scanning calorimetry

FIGS. 8A-8B show equilibrium binding isotherms 3000 nM FcRN 3× dilutionseries over 3000 RUs. FIG. 8A shows Albumin and FIG. 8B showsheteromultimer scaffold ABH1

FIG. 9 shows scheme for multivalent Albumin based heteromultimerscomprising anti-Her2/neu and anti-CD16 scFv bioactive fusions

FIGS. 10A-10B contain a non-reducing SDS PAGE analysis of theheteromultimer ABH2 fusions described in table 8. The gel indicates allconstructs form the correct complex with expected MW.

FIG. 11 shows structure of Annexin molecule based on the PDB structure1MCX. The two monomers that will be derived by splitting the Annexinmolecule are color coded as light and dark grey units. The sites offusion for the cargo protein are represented as spheres.

FIG. 12 shows a plot of the buried solvent accessible surface area atthe interface of Annexin based transporter polypeptide-1, and Annexinbased transporter polypeptide-2.

FIG. 13 shows structure of transferrin molecule based on the PDBstructure 1H76. The two monomers derived by splitting the transferrinmolecule are color coded as light and dark grey units. The sites offusion for the cargo protein are represented as spheres.

FIG. 14 shows a plot of the buried solvent accessible surface area atthe interface of two transferrin based transporter polypeptidesdescribed herein. A split transferrin near residue position 330 asdesigned herein, forms a heterodimer with about 1800 Å² of buriedsurface area.

FIG. 15 shows sequences of multimers comprising transporter polypeptidesbased on human serum albumin.

DETAILED DESCRIPTION

In the realm of therapeutic proteins, bispecific molecules exhibit dualtarget specificities or are able to simultaneously perform multiplefunctional roles by providing the necessary spatiotemporal organizationnecessary for drug action. In one aspect, bispecific molecules areparticularly interesting when the mode of therapeutic action involvesretargeting of effector cells or molecules to a target such as a tumorcell [Muller D. and Kontermann R. E. (2010) Biodrugs 24, 89-98]. Thedevelopment of bispecific therapeutic proteins with favorablepharmacokinetics and functional activity in stable and homogeneouscondition has been a challenge. Attempts have been made to assemblebispecific units from multiple antigen binding domains using a number ofapproaches. These techniques have involved using heterodimeric antibodyIgG molecule, using leucine zipper proteins such as the Fos/Jun pair orother scaffolds assembled from the alternate organizations of the lightand heavy chains of the variable domains in an antibody. Kipriyanov andLe Gall have reviewed the design of a variety of bispecific constructs[Kipriyanov S. M. & Le Gall F. (2004) Curr Opin Drug Discov Dev 7,233-242]. The use of a heterodimeric antibody IgG molecule whereinmutations are introduced in the CH3 domain of the antibody to achievethe heterodimer and hence introduce the two unique antigen binding sitesinto one molecule is very attractive because of the naturalimmunoglobulin like structure of this construct. Further, the Fc portionof the antibody is involved in interactions with the neonatal Fcreceptor (FcRn) which mediates an endocytic salvage pathway and this isattributed to improved serum half-life of the antibody molecule[Roopenian D. & Akilesh S. (2007) Nature Rev Immunol 7, 715-725]. On theother hand, antibody based bispecific molecules have been problematic inclinical trials because of the strong cytokine responses as a result ofthe concurrent effector activity induced via the Fc portion of thebispecific antibody [Weiner L. M.; Alpaugh R. K. et al. (1996) CancerImmunol Immunother 42, 141-150]. This highlights the needs for novelscaffolds that can aid in the design of bispecific and immunoconjugatemolecules.

The human serum album (HSA) protein is the most abundant component ofblood, accounting for close to 60% of the total protein in blood serumat a concentration of about 40 mg/ml. Albumin is also one of thelongest-lived proteins in the circulatory system with a half-life ofabout 19 days. Interestingly, the same endocytic salvage pathwaydependent on FcRn molecules that prevents antibody degradation is knownto interact with the HSA molecule as well [Chaudhary C.; Mehnaz S. etal. (2003) J Exp Med 197, 315-322].

HSA (shown in FIG. 1) is a non-glycosylated 585-residue singlepolypeptide protein and the 3-dimensional structure of the protein wasfirst observed using X-ray crystallography by Carter and coworkers[reviewed in Carter, D. C. & Ho, J. X. (1994) Adv Prot Chem 45,153-203]. The HSA protein consists of three homologous domains: DI, DII,DIII, attributed to gene duplication, a feature common to the serumalbumin in other species as well [Gray J. E. & Doolittle R. F. (1992)Protein Sci 1, 289-302]. Each of the three domains have been expressedand characterized separately and shown to be independently stable[Dockal M., Carter D. C. & Ruker F. (1999) J Biol Chem 274,29303-29310]. Each domain is made up of 10 helical segments and based onthe inter-helical organization each domain can be further classifiedinto 2 sub-domains comprised of helix 1-6 and 7-10 respectively. HSA has17 disulphide bonds in total and all these cysteine pairs forming thelinkages are within the individual domains. In general, HSA is a verystable due to the large number of disulphide bonds as well as thepredominantly helical fold. The sequence identities of albumin moleculesacross a number of species is quite large, greater than 70% amongalbumin cDNA derived from humans, horse, bovine, rat, etc. [Carter, D.C. & Ho, J. X. (1994) Adv Prot Chem 45, 153-203].

Split protein pairs have been used as sensors to understandprotein-protein interactions in the area of functional proteomics. Theapproach involves identifying suitable segments from a protein that canreconstitute to form an active native-like protein. Generating new splitproteins is technically demanding. For a protein to be split in afunctionally useful manner, the segmentation site has to yield twosegments that efficiently reconstitute into the quasi-native proteinwhen associated to each other. Further, the component protein segmentsshould be soluble enough to stay in solution and selectively associatewith the partner segments such that manufacture yields and purificationwill be economical. Deriving split protein segments that would recombineto form the quasi-native structure is quite challenging [Tafelmeyer P.,Johnsson N. & Johnsson K. Chem & Biol 11, 681-689]. Such split proteinshave not been used in the design of protein therapeutics, or as cargodelivery vehicles in the past.

Definitions

It is to be understood that this invention is not limited to theparticular protocols; cell lines, constructs, and reagents describedherein and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “HSA”, “HA”, “albumin”,“human serum albumin” and various capitalized, hyphenated andunhyphenated forms is a reference to one or more such proteins andincludes variants, derivatives, fragments, equivalents thereof known tothose of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

A “heteromultimer” or “heteromultimeric polypeptide” is a moleculecomprising at least a first monomer comprising a first transporterpolypeptide and a second monomer comprising a second transporterpolypeptide, wherein the second polypeptide differs in amino acidsequence from the first polypeptide by at least one amino acid residue.The heteromultimer can comprise a “heterodimer” formed by the first andsecond transporter polypeptides. In certain embodiments, theheteromultimer can form higher order tertiary structures such as, butnot restricted to trimers and tetramers. In some embodiments,transporter polypeptides in addition to the first and second transporterpolypeptides are present. In certain embodiments, the assembly oftransporter polypeptides to form the heteromultimer is driven by surfacearea burial. In some embodiments, the transporter polypeptides interactwith each other by means of electrostatic interactions and/orsalt-bridge interactions that drive heteromultimer formation by favoringheteromultimer formation and/or disfavoring homomultimer formation. Insome embodiments, the transporter polypeptides interact with each otherby means of hydrophobic interactions that drive heteromultimer formationby favoring heteromultimer formation and/or disfavoring homomultimerformation. In certain embodiments, the transporter polypeptides interactwith each other by means of covalent bond formation. In certainembodiments, the covalent bonds are formed between naturally present orintroduced cysteines that drive heteromultimer formation. In certainembodiments of the heteromultimers described herein, no covalent bondsare formed between the monomers. In some embodiments, the transporterpolypeptides interact with each other by means ofpacking/size-complementarity/knobs-into-holes/protruberance-cavity typeinteractions that drive heteromultimer formation by favoringheteromultimer formation and/or disfavoring homomultimer formation. Insome embodiments, the transporter polypeptides interact with each otherby means of cation-pi interactions that drive heteromultimer formationby favoring heteromultimer formation and/or disfavoring homomultimerformation. In certain embodiments the individual transporterpolypeptides cannot exist as isolated monomers in solution. In certainembodiments, the heteromultimer is the preferred state of the individualtransporter polypeptides as compared to the monomer.

The term “bispecific” is intended to include any agent, e.g.,heteromultimer, monomer, protein, peptide, or protein or peptidecomplex, which has two different binding specificities. For example, insome embodiments, the molecule may bind to, or interact with, (a) a cellsurface target molecule and (b) an Fc receptor on the surface of aneffector cell. In certain embodiments of a heteromultimer describedherein, at least one monomer is bispecific formed by attaching to thesame transporter polypeptide, two cargo molecules with different bindingspecificities. In certain embodiments of a heteromultimer describedherein, the heteromultimer is itself bispecific formed by attaching tothe transporter polypeptides, at least two cargo molecules withdifferent specificities. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with, (a) a cell surface target molecule such as but notlimited to cell surface antigens, (b) an Fc receptor on the surface ofan effector cell, and (c) at least one other component. Accordingly,embodiments of the heteromultimers described herein, are inclusive of,but not limited to, bispecific, trispecific, tetraspecific, and othermultispecific molecules. In certain embodiments, these molecules aredirected to cell surface antigens, such as CD30, and to other targets,such as Fc receptors on effector cells.

Unless indicated otherwise, the expression “multivalent” is usedthroughout this specification to denote a heteromultimer comprising atleast two sites of attachment for target molecules. The multivalentheteromultimer is designed to have multiple binding sites for desiredtargets. In certain embodiments, the binding sites are on at least onecargo molecules attached to a transporter polypeptide. In certainembodiments, at least one binding site is on a transporter polypeptide.The expression “bivalent” is used throughout this specification todenote a heteromultimer comprising two target binding sites. In certainembodiments of a bivalent heteromultimer, both binding sites are on thesame monomer. The expression “trivalent” is used throughout thisspecification to denote a heteromultimer comprising three target bindingsites. The expression “tetravalent” is used throughout thisspecification to denote a heteromultimer comprising four target bindingsites.

“Fusion proteins” and polypeptides are created by joining two or moregenes that originally code for separate polypeptides. Translation ofthis fusion gene results in a single polypeptide with functionalproperties derived from each of the original polypeptides. Inembodiments of the heteromultimers described herein, at least onemonomer may comprise a fusion protein formed by the fusion of at leastone cargo polypeptide to the N- or C-terminus of a transporterpolypeptide.

The term “substantially purified” refers to a heteromultimer describedherein, or variant thereof that may be substantially or essentially freeof components that normally accompany or interact with the protein asfound in its naturally occurring environment, i.e. a native cell, orhost cell in the case of recombinantly produced heteromultimer that incertain embodiments, is substantially free of cellular material includespreparations of protein having less than about 30%, less than about 25%,less than about 20%, less than about 15%, less than about 10%, less thanabout 5%, less than about 4%, less than about 3%, less than about 2%, orless than about 1% (by dry weight) of contaminating protein. When theheteromultimer or variant thereof is recombinantly produced by the hostcells, the protein in certain embodiments is present at about 30%, about25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%,about 2%, or about 1% or less of the dry weight of the cells. When theheteromultimer or variant thereof is recombinantly produced by the hostcells, the protein, in certain embodiments, is present in the culturemedium at about 5 g/L, about 4 g/L, about 3 g/L, about 2 g/L, about 1g/L, about 750 mg/L, about 500 mg/L, about 250 mg/L, about 100 mg/L,about 50 mg/L, about 10 mg/L, or about 1 mg/L or less of the dry weightof the cells. In certain embodiments, “substantially purified”heteromultimer produced by the methods described herein, has a puritylevel of at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, specifically, a puritylevel of at least about 75%, 80%, 85%, and more specifically, a puritylevel of at least about 90%, a purity level of at least about 95%, apurity level of at least about 99% or greater as determined byappropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, andcapillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe protein has been secreted, including medium either before or after aproliferation step. The term also may encompass buffers or reagents thatcontain host cell lysates, such as in the case where a heteromultimerdescribed herein is produced intracellularly and the host cells arelysed or disrupted to release the heteromultimer.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two monomeric polypeptideswhich interact with each other and result in the transformation ofunfolded or improperly folded polypeptides to native, properly foldedpolypeptides.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a cargo polypeptide thatis comprised by a heteromultimer described herein relative to its nativeform. Serum half-life is measured by taking blood samples at varioustime points after administration of heteromultimer, and determining theconcentration of that molecule in each sample. Correlation of the serumconcentration with time allows calculation of the serum half-life.Increased serum half-life desirably has at least about two-fold, but asmaller increase may be useful, for example where it enables asatisfactory dosing regimen or avoids a toxic effect. In someembodiments, the increase is at least about three-fold, at least aboutfive-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of a cargo polypeptide comprised by a heteromultimerdescribed herein, relative to its non-modified form. Therapeutichalf-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule or an increase or decrease in receptor-mediatedclearance of the molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoramidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, praline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as β-alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, α-methyl amino acids (e.g. α-methyl alanine),D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine,β-hydroxy-histidine, homohistidine), amino acids having an extramethylene in the side chain (“homo” amino acids), and amino acids inwhich a carboxylic acid functional group in the side chain is replacedwith a sulfonic acid group (e.g., cysteic acid). The incorporation ofnon-natural amino acids, including synthetic non-native amino acids,substituted amino acids, or one or more D-amino acids into the proteinsof the present invention may be advantageous in a number of differentways. D-amino acid-containing peptides, etc., exhibit increasedstability in vitro or in vivo compared to L-amino acid-containingcounterparts. Thus, the construction of peptides, etc., incorporatingD-amino acids can be particularly useful when greater intracellularstability is desired or required. More specifically, D-peptides, etc.,are resistant to endogenous peptidases and proteases, thereby providingimproved bioavailability of the molecule, and prolonged lifetimes invivo when such properties are desirable. Additionally, D-peptides, etc.,cannot be processed efficiently for major histocompatibility complexclass II-restricted presentation to T helper cells, and are therefore,less likely to induce humoral immune responses in the whole organism.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and [0139] 8) Cysteine (C),    Methionine (M) (see, e.g., Creighton, Proteins: Structures and    Molecular Properties (W H Freeman & Co.; 2nd edition (Dec. 1993)

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide. A polynucleotide encoding apolypeptide of the present invention, including homologs from speciesother than human, may be obtained by a process comprising the steps ofscreening a library under stringent hybridization conditions with alabeled probe having a polynucleotide sequence of the invention or afragment thereof, and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, or other nucleic acids, or combinationsthereof under conditions of low ionic strength and high temperature asis known in the art. Typically, under stringent conditions a probe willhybridize to its target subsequence in a complex mixture of nucleic acid(including but not limited to, total cellular or library DNA or RNA) butdoes not hybridize to other sequences in the complex mixture. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “prokaryote” refers to prokaryotic organisms.For example, a non-eukaryotic organism can belong to the Eubacteria(including but not limited to, Escherichia coli, Thermus thermophilus,Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonasaeruginosa, Pseudomonas putida, etc.) phylogenetic domain, or theArchaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment. An animal may be acompanion animal (e.g., dogs, cats, and the like), farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like).

The term “effective amount” as used herein refers to that amount ofheteromultimer being administered, which will relieve to some extent oneor more of the symptoms of the disease, condition or disorder beingtreated. Compositions containing the heteromultimer described herein canbe administered for prophylactic, enhancing, and/or therapeutictreatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

The term “segmentation” refers to a precise internal splice of theoriginal protein sequence which results in “segments” of the proteinsequence that preferentially associate as heteromultimers to form aquasi-protein.

Quasi-Native Structure:

With reference to a native protein or its structure, quasi-nativeproteins and/or ‘quasi-native structures’ present the native proteinlike functional and structural characteristics. Proteins are naturallydynamics molecules and display an ensemble of structural configurationsalthough we ascribe a native structure to it, such as the one obtainedby X-ray crystallography. The alternate structural configurationsobserved in the ensemble of geometries of that protein can be deemed tobe quasi-native structures relative to each other or relative to thestructure observed in the crystal. On a different front, homologousproteins sequences or proteins belonging to common structural familiestend to fold into similar structural geometries. The member proteinsbelonging to this family can be deemed to achieve a quasi-nativestructure relative to each other. Some of the unique sequences in theprotein family could also exhibit similar functional attributes andhence can be referred to as quasi-native proteins relative to eachother. In the case of heteromultimers described here comprising of twoor more monomeric proteins each of which have a transporter polypeptidecomponent, the transporter polypeptides assemble to form a quasi-nativestructure. The reference native protein in this case is the protein fromwhich the transporter polypeptide is derived and the reference nativestructure is the structure of the protein from which the transporterpolypeptide is derived. We describe a case where two or more differentpolypeptides self-assemble to form a heteromultimeric structural andexhibit functional characteristics like a native protein which itself isa monomeric entity. In certain embodiments, we present polypeptidesegments derived from albumin that self-assemble to form aheteromultimer that exhibits native albumin like functionalcharacteristics such as FcRn binding and structural characteristics. Incertain embodiments, we present polypeptide segments derived fromtransferrin that self-assemble to form a heteromultimer that exhibitsnative transferrin like structural and functional characteristics. Incertain embodiments, we present polypeptide segments derived fromannexin that self-assemble to form a heteromultimer that exhibits nativeannexin like structural and functional characteristics. Theseheteromultimers are referred to as being quasi-native.

Transporter Polypeptide

As used herein, the term “transporter polypeptide” or “transporterpolypeptide” or “transporter peptide” or “transporter” refers to apolypeptide, such that said transporter polypeptide is capable offorming heteromultimeric proteins with other such transporterpolypeptides in solution, and wherein said heteromultimeric proteinshave a quasi-native structure of a monomeric protein from which at leastone transporter polypeptide is derived. In certain embodiments of theheteromultimers described herein, all transporter polypeptides arederived from the same albumin or alloalbumin protein. In certain otherembodiments, the heteromultimers are formed by transporter polypeptidesderived from various albumin and alloalbumin proteins. In certainembodiments of the heteromultimers described herein, the transporterpolypeptides are derived from transferrin. In certain embodiments of theheteromultimers described herein, all transporter polypeptides arederived from annexin proteins. In certain embodiments, theheteromultimers are formed by transporter polypeptides derived from thesame annexin protein. In some embodiments, the heteromultimers areformed by transporter polypeptides derived from different annexinproteins. In an embodiment, the heteromultimers are formed bytransporter polypeptides derived from annexin A2.

In certain embodiments, transporter polypeptides are segments of a wholeprotein, wherein said segments are capable of assembling to form aheteromultimer. In certain embodiments, the transporter polypeptides aresegments derived from a coiled coil protein. In certain embodiments, thetransporter polypeptides are segments derived from a leucine-zipperprotein. In an embodiment, the transporter polypeptides are segmentsfrom a beta-barrel protein. In an embodiment, transporter polypeptidesare segments obtained from a beta-propeller protein. In someembodiments, the transporter polypeptides are segments obtained from ahelical bundle protein. In embodiments, the transporter polypeptides aregenerated from for instance, but not restricted to proteins comprising azinc finger motif, a helix-turn-helix motif or a beta-hairpin motif. Insome embodiments, the transporter polypeptides are segments obtainedfrom non-immunogenic proteins that are structurally stable, and havefavorable biological properties.

Albumin

As used herein, “albumin” refers collectively to albumin protein oramino acid sequence, or an albumin segment or variant, having one ormore functional activities (e.g., biological activities) of albumin. Inparticular “albumin” refers to human albumin or segments thereof (seefor example, EP 201 239, EP 322 094 WO 97/24445, WO95/23857) especiallythe mature form of human albumin as shown in FIG. 1, or albumin fromother vertebrates, or segments thereof, or analogs or variants of thesemolecules or fragments thereof. In certain embodiments, albumin refersto a truncated version of albumin.

The term “quasi-albumin” refers to a heteromultimer molecule that hasstructure and/or function similar to the whole albumin, and wherein saidheteromultimer molecule is formed by the assembly of two or moremonomeric polypeptides designed based on the sequence of the wholealbumin. In certain embodiments, the monomeric polypeptides are“segments” that preferentially associate as heteromultimeric pairs toform a quasi-protein. In some embodiments, the quasi-albumin has 90% ofthe activity of the whole albumin. In some embodiments, thequasi-albumin has 75% of the activity of whole-albumin. In anembodiment, the quasi-albumin has 50% of the activity of whole albumin.In some embodiments, the quasi-albumin has 50-75% of the activity ofwhole albumin. In an embodiment, quasi-albumin has 80% of the activityof whole albumin. In some embodiments, the quasi-albumin has 90% of thestructure of whole albumin as determined by molecular modeling. In someembodiments, the quasi-albumin has 80% of the structure of whole albuminas determined by molecular modeling. In some embodiments, thequasi-albumin has 70% of the structure of whole albumin as determined bymolecular modeling. In some embodiments, the quasi-albumin has 50% ofthe structure of whole albumin as determined by molecular modeling. Insome embodiments, the quasi-albumin has 50%-75% of the structure ofwhole albumin as determined by molecular modeling.

The terms, human serum albumin (HSA) and human albumin (HA) are usedinterchangeably herein. The terms, “albumin and serum albumin” arebroader, and encompass human serum albumin (and fragments and variantsthereof) as well as albumin from other species (and fragments andvariants thereof).

In certain embodiments, each albumin-based monomer of theheteromultimeric proteins described herein is based on a variant ofnormal HA. Each cargo polypeptide portion of the heteromultimericproteins of the invention may also be variants of the Therapeuticproteins as described herein. The term “variants” includes insertions,deletions and substitutions, either conservative or non conservative,where such changes do not substantially alter one or more of theoncotic, useful ligand-binding and non-immunogenic properties ofalbumin, or the active site, or active domain which confers thetherapeutic activities of the Therapeutic proteins.

In certain embodiments, the heteromultimeric proteins described hereininclude naturally occurring polymorphic variants of human albumin andfragments of human albumin, for example those fragments disclosed in EP322 094 (namely HA (Pn), where n is 369 to 419).

In certain embodiments, the albumin is derived from any vertebrate,especially any mammal that includes but is not limited to human, cow,sheep, rat, mouse, rabbit, horse, dog or pig. In certain embodiments,the albumin is derived from non-mammalian albumins including, but arenot limited to hen and salmon.

The sequence of human albumin is as shown:

SEQ ID NO: 1 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKK LVAASQAALGL

Alloalbumin

An alloalbumin is a genetic variant of albumin. In certain embodimentsthe alloalbumin is human alloalbumin (HAA). Alloalbumins that differ inelectrophoretic mobility from albumin have been identified throughpopulation genetics surveys in the course of clinical electrophoresis,or in blood donor surveys. As markers of mutation and migration,alloalbumins are of interest to geneticists, biochemists, andanthropologists, but most of these alloalbumin are not associated withdisease (Minchioti et al. Human Mutations 29(8), 1007-1016(2008)).

TABLE 1 List of substitutions comprised by various alloalbumins ascompared to HA of SEQ ID NO: 1. Thermostability, half- life informationand other HAAs are provided in Krogh- hansen et al. Biochim Biophys Acta1747, 81-88(2005); and WO2011051489 incorporated by reference herein.Thermostability (C.) Effect on (positive = stabilizing, half-lifeMutation negative = destabilizing) (% change) H3Y N/A N/A H3Q N/A N/AQ32Stop N/A N/A E60K N/A N/A D63N 6.07 N/A L66P N/A N/A E82K 2.03 N/AR114G N/A N/A R114Stop N/A N/A E119K N/A N/A V122E 0.57 N/A H128R N/AN/A Y140C N/A N/A A175Stop N/A N/A C177F −1.59  N/A R218H N/A N/A R218PN/A N/A K225Q* −4.86  N/A K240E N/A N/A E244Stop N/A N/A Q268R N/A N/AD269G 3.67 N/A K276N 4.87 N/A K313N −7.16  N/A D314G −0.38  N/A D314VN/A N/A N318K N/A N/A A320T, & -1R N/A 6.16 E321K 1.42 N/A E333K −2.56 N/A E354K N/A N/A E358K N/A N/A K359K −6.56  N/A D365H 0.89 N/A D365VN/A N/A E368G N/A N/A K372E N/A N/A D375N N/A N/A D375H −0.09  N/A E376KN/A N/A E376Q N/A N/A E382K N/A N/A Q385Stop N/A N/A Y401Stop N/A N/AR410C N/A N/A E479K N/A N/A D494N N/A 0.84 E501K 0.13 N/A E505K 1.87 N/AI513N N/A N/A V533M N/A N/A K536E N/A N/A K541E 6.12 N/A D550G N/A N/AD550A N/A N/A K560E 0.70 N/A D563N 4.17 N/A E565K N/A N/A E570K −6.53 N/A K573E 2.08 2.7  K574N N/A N/A L575insertion(TCCCKSSCLR −5.30  N/ALITSHLKASQPTMRIRERK) Frameshift after 567; Stop N/A −5.7%  at 582Frameshift after 572; Stop N/A −8.9%  at 578

Annexin:

As used herein, “annexin” refers to a group of cellular proteins foundin eukaryotic organisms. Annexin is also known as lipocortin. As usedherein “annexin” may refer to any annexin protein, or to specificannexin proteins such as “annexin A1,” “annexin A2,” and “annexin A5.”Annexins are characterized by their calcium dependent ability to bindnegatively charged phospholipids (i.e. membrane walls). Annexins arecharacterized by a repeat protein scaffold limited to 30-50 kDa in sizewith fairly ubiquitous tissue distribution. The basic structure of anannexin is composed of two domains: a structurally conserved C terminal“core” region and a divergent N terminal domain. The core region bindsthe phospholipid cellular membrane in a Ca²⁺ dependent manner. The Nterminal region binds cytoplasmic proteins. Annexins are important invarious cellular and physiological processes and provide a membranescaffold. The C terminal core is composed of four annexin repeats.Annexin is characterized by its flexible repeat-like nature thatinfluences its intrinsic membrane-sensing abilities. For instance, theaffinity towards specific biomembranes can be controlled by the numberof repeats. With the characteristic phospholipid sensing, annexin can beuseful to sense/target intestinal junctions for drug delivery. Anotherpotential application for an annexin is targeting intestinal tightjunctions and the Zonula Occludens region (ZO-1), which is known to beparticularly difficult to traverse for larger protein therapeutics,significantly impairing drug absorption.

The term “quasi-annexin” refers to a heteromultimer molecule that hasstructure and/or function similar to the whole annexin, and wherein saidheteromultimer molecule is formed by the assembly of two or moremonomeric polypeptides designed based on the sequence of the wholeannexin. In certain embodiments, the monomeric polypeptides are“segments” that preferentially associate as heteromultimeric pairs toform a quasi-protein. In some embodiments, the quasi-annexin has 90% ofthe activity of the whole annexin. In some embodiments, thequasi-annexin has 75% of the activity of whole-annexin. In anembodiment, the quasi-annexin has 50% of the activity of whole annexin.In some embodiments, the quasi-annexin has 50-75% of the activity ofwhole annexin. In an embodiment, quasi-annexin has 80% of the activityof whole annexin. In some embodiments, the quasi-annexin has 90% of thestructure of whole annexin as determined by molecular modeling. In someembodiments, the quasi-annexin has 80% of the structure of whole annexinas determined by molecular modeling. In some embodiments, thequasi-annexin has 70% of the structure of whole annexin as determined bymolecular modeling. In some embodiments, the quasi-annexin has 50% ofthe structure of whole annexin as determined by molecular modeling. Insome embodiments, the quasi-annexin has 50%-75% of the structure ofwhole annexin as determined by molecular modeling.

The sequence of Human wild-type Annexin A2 is as shown:

SEQ ID NO: 14 GSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLAKGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN

Transferrin:

Transferrins are monomeric proteins of about 76 kDa molecular weightpresent in all vertebrates and function as a iron-binding andtransporting protein. Recombinant human transferrin and its fusions isbeing considered for the management of various diseases includingthalassemia, atransferrinemia, age related macular degeneration, type 2diabetes, during stem cell transplantation and in the treatment of acuteinfectious disease caused by the anthrax bacteria. Transferrin is stablein the gastrointestinal environment and a number of studies have shownthat intact protein-transferrin conjugates can be orally delivered andremain bioactive.

The term “quasi-transferrin” refers to a heteromultimer molecule thathas structure and/or function similar to the whole transferrin, andwherein said heteromultimer molecule is formed by the assembly of two ormore monomeric polypeptides designed based on the sequence of the wholetransferrin. In certain embodiments, the monomeric polypeptides are“segments” that preferentially associate as heteromultimeric pairs toform a quasi-protein. In some embodiments, the quasi-transferrin has 90%of the activity of the whole transferrin. In some embodiments, thequasi-transferrin has 75% of the activity of whole-transferrin. In anembodiment, the quasi-transferrin has 50% of the activity of wholetransferrin. In some embodiments, the quasi-transferrin has 50-75% ofthe activity of whole transferrin. In an embodiment, quasi-transferrinhas 80% of the activity of whole transferrin. In some embodiments, thequasi-transferrin has 90% of the structure of whole transferrin asdetermined by molecular modeling. In some embodiments, thequasi-transferrin has 80% of the structure of whole transferrin asdetermined by molecular modeling. In some embodiments, thequasi-transferrin has 70% of the structure of whole transferrin asdetermined by molecular modeling. In some embodiments, thequasi-transferrin has 50% of the structure of whole transferrin asdetermined by molecular modeling. In some embodiments, thequasi-transferrin has 50%-75% of the structure of whole transferrin asdetermined by molecular modeling.

The sequence of wildtype Human Transferrin is as shown:

SEQ ID NO: 19 MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQSFRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVTLDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVVKKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPEPRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCSTLNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRDQYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKEDLIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHGFLKVPPRMDA KMYLGYEYVT AIRNLREGTC PEAPTDECKPVKWCALSHHE RLKCDEWSVN SVGKIECVSA ETTEDCIAKIMNGEADAMSL DGGFVYIAGK CGLVPVLAEN YNKSDNCEDTPEAGYFAVAV VKKSASDLTW DNLKGKKSCH TAVGRTAGWNIPMGLLYNKI NHCRFDEFFS EGCAPGSKKD SSLCKLCMGSGLNLCEPNNK EGYYGYTGAF RCLVEKGDVA FVKHQTVPQNTGGKNPDPWA KNLNEKDYEL LCLDGTRKPV EEYANCHLARAPNHAVVTRK DKEACVHKIL RQQQHLFGSN VTDCSGNFCLFRSETKDLLF RDDTVCLAKL HDRNTYEKYL GEEYVKAVGN LRKCSTSSLL EACTFRRP

Cargo Molecule:

A heteromultimer described herein comprises monomers that comprise atleast one cargo molecule, and at least one transporter polypeptide, saidcargo molecule and transporter polypeptide associated with one another,by means inclusive of, but not restricted to genetic fusion or chemicalconjugation. In certain embodiments, at least one cargo molecule is atherapeutic agent. In certain agents, the cargo molecule is a toxin. Incertain embodiments, the cargo molecule is an antigen, or analogsthereof. In an embodiment, the cargo molecule is a natural product,analog, or prodrug thereof. In certain embodiments, the cargo moleculeis a therapeutic agent such as a cytotoxin, e.g., a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6mercaptopurine, 6thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

In certain embodiment, the cargo molecule is a biomolecule. In anembodiment, the cargo molecule is a natural or synthetic nucleic acid.In some embodiments, at least one cargo molecule is one or more of aDNA, PNA, and/or RNA oligomer. In certain embodiments, a heteromultimerdescribed herein comprises monomeric proteins that comprise at least onecargo polypeptide, or fragments or variants thereof, and at least onetransporter polypeptide, said cargo polypeptide and transporterpolypeptide associated with one another, by means inclusive of, but notrestricted to genetic fusion or chemical conjugation

As used herein, “Cargo polypeptide” refers to proteins, polypeptides,antibodies, peptides or fragments or variants thereof, having one ormore therapeutic and/or biological activities. Cargo polypeptidesencompassed by the invention include but are not limited to, proteins,polypeptides, peptides, antibodies, substrates or ligands totherapeutically relevant target proteins and biologics. (The termspeptides, proteins, and polypeptides are used interchangeably herein.)Specifically the term “Cargo polypeptide” encompasses antibodies andfragments and variants thereof. Thus a heteromultimer described hereinmay contain at least a fragment or variant of a cargo polypeptide,and/or at least a fragment or variant of an antibody. Additionally, incertain embodiments, the term “Cargo polypeptide” refers to theendogenous or naturally occurring correlate of a cargo polypeptide.

As a non-limiting example, a “Cargo biomolecule” is a biomolecule suchas but not restricted to a protein, DNA, or RNA that is useful to treat,prevent or ameliorate a disease, condition or disorder. As anon-limiting example, a “Cargo polypeptide” may be one that bindsspecifically to a particular cell type (normal (e.g., lymphocytes) orabnormal e.g., (cancer cells)) and therefore may be used to target acompound (drug, or cytotoxic agent) to that cell type specifically.

In another non-limiting example, a “Cargo molecule” is a molecule thathas a biological, activity, and in particular, a biological activitythat is useful for treating preventing or ameliorating a disease. Anon-inclusive list of biological activities that may be possessed by aCargo molecule, for instance a Cargo polypeptide includes, enhancing theimmune response, promoting angiogenesis, inhibiting angiogenesis,regulating hematopoietic functions, stimulating nerve growth, enhancingan immune response, inhibiting an immune response, or any one or more ofthe biological activities described herein.

Cargo polypeptides corresponding to a cargo polypeptide portion of aheteromultimer protein described herein, such as cell surface andsecretory proteins, are often modified, by the attachment of one or moreoligosaccharide groups. The modification, referred to as glycosylation,can dramatically affect the physical properties of proteins and can beimportant in protein stability, secretion, and localization.Glycosylation occurs at specific locations along the polypeptidebackbone. There are usually two major types of glycosylation:glycosylation characterized by O-linked oligosaccharides, which areattached to serine or threonine residues; and glycosylationcharacterized by N-linked oligosaccharides, which are attached toasparagine residues in an Asn-X-Ser/Thr sequence, where X can be anyamino acid except proline. N-acetylneuramic acid (also known as sialicacid) is usually the terminal residue of both N-linked and Blinkedoligosaccharides. Variables such as protein structure and cell typeinfluence the number and nature of the carbohydrate units within thechains at different glycosylation sites. Glycosylation isomers are alsocommon at the same site within a given cell type.

Table 2 provides a non-exhaustive list of Cargo polypeptides thatcorrespond to a Cargo polypeptide portion of a heteromultimer describedherein. The “Cargo Polypeptide” column discloses Cargo polypeptidemolecules followed by parentheses containing scientific and brand namesthat comprise, or alternatively consist of, that Cargo polypeptidemolecule or a fragment or variant thereof. In an embodiment the cargomolecule is a molecule that binds to a protein disclosed in the “Cargopolypeptide” column, or in Zhu et al. (Nucleic Acids Res. 38(1),D787-D791 (2009)); Wishart et al. (Nucleic Acids Res 36, D901-D906(2008)); Ahmed et al. (Nucleic Acids Res 39, D960-D967 (2011))incorporated by reference herein, or a protein that belongs in the classof therapeutic target molecules.

“Cargo polypeptide” as used herein may refer either to an individualCargo polypeptide molecule (as defined by the amino acid sequenceobtainable from the CAS and Genbank accession numbers), or to the entiregroup of Cargo polypeptide associated with a given Cargo polypeptidemolecule disclosed in this column, or a Cargo polypeptide that binds toa polypeptide molecule disclosed in this column.

TABLE 2 Non-exhaustive list of Cargo polypeptides that correspond to aCargo polypeptide portion of a heteromultimer Cargo PolypeptideBiological Activity Exemplary Activity Assay Indication EPO(Erythropoietin; Stimulates cellular Cell proliferation assay Anemia;Anemia in Renal Disease; Epoetin alfa; Epoetin differentiation of bone-using a erythroleukemic Anemia in Oncology Patients; beta;Gene-activated marrow stem cells at an cell line TF-1. (KitamuraBleeding Disorders; Chronic Renal erythropoietin; early stage of et al.1989 J. Cell. Failure; Chronic Renal Failure in Pre- Darbepoetin- alpha;etythropoiesis; Physiol. 140: 323) Dialysis Patients; Renal Disease;NESP; Epogen; Procrit; accelerates the End-Stage Renal Disease;End-Stage Eprex; Erypo; Espo; proliferation and Renal Disease inDialysis Patients; Epoimmun; EPOGIN; maturation of Chemotherapy;Chemotherapy in NEORECORMON; terminally Cancer Patients; Anemia inHEMOLINK; Dynepo; differentiating cells into zidovudine-treated HIVpatients; ARANESP) erythrocytes; and Anemia in zidovudine-treatedmodulates the level of patients; Anemia in HIV patients; circulatingerythrocytes. Anemia in premature infants; Surgical patients (pre and/orpost surgery); Surgical patients (pre and/or post surgery) who areanemic; Surgical patients (pre and/or post surgery) who are undergoingelective surgery; Surgical patients (pre and/or post surgery) who areundergoing elective, non-cardiac surgery; Surgical patients (pre and/orpost surgery) who are undergoing elective, non-cardiac, non-vascularsurgery; Surgical patients (pre and/or post surgery) who are undergoingelective, non-vascular surgery; Surgical patients (pre and/or postsurgery) who are undergoing cardiac and/or vascular surgery; Aplasticanemia; Refractory anemia; Anemia in Inflammatory Bowel Disease;Refractory anemia in Inflammatory Bowel Disease; Transfusion avoidance;Transfusion avoidance for surgical patients; Transfusion avoidance forelective surgical patients; Transfusion avoidance for electiveorthopedic surgical patients; Patients who want to Increase Red BloodCells. G-CSF (Granulocyte Stimulates the Proliferation of murineChemoprotection; Adjunct to colony-stimulating proliferation and NFS-60cells (Weinstein Chemotherapy; Inflammatory factor; Granulokine;differentiation of the et al, Proc Natl Acad Sci disorders; Cancer;Leukemia; KRN 8601; Filgrastim; progenitor cells for USA 1986; 83,pp5010-4) Myelocytic leukemia; Neutropenia, Lenograstim; granulocytesand Primary neutropenias (e.g.; Meograstim; monocytes- Kostmannsyndrome); Secondary Nartograstim; macrophages. neutropenia; Preventionof Neupogen; NOPIA; neutropenia; Prevention and Gran; GRANOCYTE;treatment of neutropenia in HIV- Granulokine; Neutrogin; infectedpatients; Prevention and Neu-up; Neutromax) treatment of neutropeniaassociated with chemotherapy; Infections associated with neutropenias;Myelopysplasia; Autoimmune disorders; Psoriasis; Mobilization ofhematopoietic progenitor cells; Wound Healing; Autoimmune Disease;Transplants; Bone marrow transplants; Acute myelogeneous leukemia;Lymphoma, Non- Hodgkin's lymphoma; Acute lymphoblastic leukemia;Hodgkin's disease; Accelerated myeloid recovery; Glycogen storagedisease. GM-CSF (Granulocyte- Regulates Colony Stimulating Bone MarrowDisorders; Bone macrophage colony- hematopoietic cell Assay: Testa, N.G., et marrow transplant; Chemoprotection; stimulating factor;differentiation, gene al., “Assays for Hepatitis C; HIV Infections;Cancer; rhuGM- CSF; BI 61012; expression, growth, and hematopoieticgrowth Lung Cancer; Melanoma; Malignant Prokine; Molgramostim; function.factors.” Balkwill F R melanoma; Mycobacterium avium Sargramostim; GM-(edt) Cytokines, A complex; Mycoses; Leukemia; CSF/IL 3 fusion;practical Approach, pp Myeloid Leukemia; Infections; Milodistim; 229-44;IRL Press Neonatal infections; Neutropenia; Leucotropin; Oxford 1991.Mucositis; Oral Mucositis; Prostate PROKINE; Cancer; Stem CellMobilization; LEUKOMAX; Vaccine Adjuvant; Ulcers (such as Interberin;Leukine; Diabetic, Venous Stasis, or Pressure Leukine Liquid; Ulcers);Prevention of neutropenia; Pixykine) Acute myelogenous leukemia;Hematopoietic progenitor cell mobilization; Lymphoma; Non- Hodgkin'slymphoma; Acute Lymphoblastic Leukemia; Hodgkin's disease; Acceleratedmyeloid recovery; Transplant Rejection; Xenotransplant Rejection. Humangrowth hormone Binds to two GHR Ba/F3-hGHR Acromegaly; Growth failure;Growth (Pegvisamont; molecules and Induces proliferation assay, ahormone replacement; Growth Somatrem; Somatropin; signal transductionnovel specific bioassay hormone deficiency; Pediatric TROVERT; throughreceptor for serum human growth Growth Hormone Deficiency; AdultPROTROPIN; BIO- dimerization hormone. J Clin Growth Hormone Deficiency;TROPIN; Endocrinol Metab 2000 Idiopathic Growth Hormone HUMATROPE;November; 85(11): Deficiency; Growth retardation; NUTROPIN; 4274-9Prader- Willi Syndrome; Prader-Willi NUTROPIN AQ; Plasma growth hormoneSyndrome in children 2 years or NUTROPHIN; (GH) immunoassay and older;Growth deficiencies; Growth NORDITROPIN; tibial bioassay, Appl failureassociated with chronic renal GENOTROPIN; Physiol 2000 December;insufficiency; Osteoporosis; SAIZEN; SEROSTIM) 89(6): Postmenopausalosteoporosis; 2174-8 Growth hormone Osteopenia, Osteoclastogenesis;(hGH) receptor mediated burns; Cachexia; Cancer Cachexia; cell mediatedDwarfism; Metabolic Disorders; proliferation, Growth Obesity; Renalfailure; Turner's Horm IGF Res Syndrome; Fibromyalgia; Fracture 2000October; treatment; Frailty, AIDS wasting; 10(5): 248-55 Muscle Wasting;Short Stature; International standard Diagnostic Agents; Female forgrowth hormone, Infertility; lipodystrophy. Horm Res 1999; 51 Suppl 1:7-12 Insulin (Human insulin; Stimulates glucose Insulin activity may beHyperglycemia; Diabetes; Diabetes Insulin aspart; Insulin uptake andpromotes assayed in vitro using a Insipidus; Diabetes mellitus; Type 1Glargine; Insulin lispro; glycogenesis and [3-H]-glucose uptakediabetes; Type 2 diabetes; Insulin Lys-B28 Pro- B29; lipogenesis. assay.(J Biol Chem 1999 resistance; Insulin deficiency; lyspro; LY 275585;Oct. 22; 274(43): 30864- Hyperlipidemia; Hyperketonemia;diarginylinsulin; Des- 30873). Non- insulin dependent Diabetes B26-B30-insulin- B25- Mellitus (NIDDM); Insulin- amide; Insulin detemir;dependent Diabetes Mellitus LABI; NOVOLIN; (IDDM); A ConditionAssociated NOVORAPID; With Diabetes Including, But Not HUMULIN; LimitedTo Obesity, Heart Disease, NOVOMIX 30; Hyperglycemia, Infections,VELOSULIN; Retinopathy, And/Or Ulcers; NOVOLOG; LANTUS; MetabolicDisorders; Immune ILETIN; HUMALOG; Disorders; Obesity; VascularMACRULIN; Disorders; Suppression of Body EXUBRA; INSUMAN; Weight;Suppression of Appetite; ORALIN; ORALGEN; Syndrome X. HUMAHALE;HUMAHALIN) Interferon alfa Confers a range of Anti-viral assay: Viralinfections; HIV Infections; (Interferon alfa-2b; cellular responsesRubinstein S, Familletti Hepatitis; Chronic Hepatitis; recombinant;Interferon including antiviral, P C, Pestka S. (1981) Hepatitis B;Chronic Hepatitis B; alfa- n1; Interferon alfa- antiproliferative,Convenient assay for Hepatitis C; Chronic Hepatitis C; n3; Peginterferonalpha- antitumor and interferons. J. Virol. Hepatitis D; ChronicHepatitis D; 2b; Ribavirin and immunomodulatory 37(2): 755-8; Anti-Human Papillomavirus; Herpes interferon alfa- 2b; activities; stimulateproliferation assay: Gao Simplex Virus Infection; External Interferonalfacon-1; production of two Y, et al (1999) Sensitivity CondylomataAcuminata; HIV; HIV interferon consensus; enzymes: a protein of anepstein-barr virus- Infection; Oncology; Cancer; Solid YM 643; CIFN;kinase and an positive tumor line, Tumors; Melanoma; Malignantinterferon- alpha oligoadenylate Daudi, to alpha interferon Melanoma;Renal Cancer (e.g., Renal consensus; recombinant synthetase. correlateswith Cell Carcinoma); Lung Cancer (e.g,. methionyl consensus expressionof a GC-rich Non-Small Cell Lung Cancer or interferon; recombinant viraltranscript. Mol Cell Small Cell Lung Cancer) Colon consensus interferon;Biol. 19(11): 7305-13. Cancer; Breast Cancer; Liver Cancer; CGP 35269;RO Prostate Cancer; Bladder Cancer; 253036; RO 258310; Gastric Cancer;Sarcoma; AIDS- INTRON A; PEG- Related Kaposi's Sarcoma; INTRON; OIF;Lymphoma; T Cell Lymphoma; OMNIFERON; PEG- Cutaneous T-Cell Lymphoma;Non- OMNIFERON; Hodgkin's Lymphoma; Brain Cancer; VELDONA; PEG- Glioma;Glioblastoma Multiforme; REBETRON; Cervical Dysplasia; Leukemia; ROFERONA; Preleukemia; Bone Marrow WELLFERON; Disorders; Bone Disorders; HairyALFERON N/LDO; Cell Leukemia; Chronic REBETRON; Myelogeonus Leukemia;ALTEMOL; Hematological Malignancies; VIRAFERON PEG; HematologicalDisorders; Multiple PEGASYS; Myeloma; Bacterial Infections; VIRAFERON;Chemoprotection; VIRAFON; Thrombocytopenia; Multiple AMPLIGEN;Sclerosis; Pulmonary Fibrosis; Age- INFERGEN; Related MacularDegeneration; INFAREX; ORAGEN) Macular Degeneration; Crohn's Disease;Neurological Disorders; Arthritis; Rheumatoid Arthritis; UlcerativeColitis; Osteoporosis, Osteopenia, Osteoclastogenesis; Fibromyalgia;Sjogren's Syndrome; Chronic Fatigue Syndrome; Fever; Hemmorhagic Fever;Viral Hemmorhagic Fevers; Hyperglycemia; Diabetes; Diabetes Insipidus;Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non- insulindependent Diabetes Mellitus (NIDDM); Insulin- dependent DiabetesMellitus (IDDM); A Condition Associated With Diabetes Including, But NotLimited To Obesity, Heart Disease, Hyperglycemia, Infections,Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;Obesity; Vascular Disorders; Suppression of Body Weight; Suppression ofAppetite; Syndrome X. Calcitonin (Salmon Regulates levels ofHypocalcemic Rat Bone Disorders; Fracture prevention; Calcitonin(Salcatonin); calcium and phosphate Bioassay, bone resorbingHypercalcemia; Malignant Calcitonin human- in serum; causes a assay andthe pit assay, hypercalcemia; Osteoporosis; Paget's salmon hybrid;reduction in serum CT receptor binding disease; Osteopenia, Forcaltonin;Fortical; calcium--an effect assay, CAMP stimulation Osteoclastogenesis;osteolysis; Calcitonin; Calcitonina opposite to that of assay: J BoneMiner Res osteomyelitis; osteonecrosis; Almirall; Calcitonina humanparathyroid 1999 August; 14(8): periodontal bone loss; osteoarthritis;Rubber; Calcimar; hormone. 1425-31 rheumatoid arthritis; osteopetrosis;Calsynar; Calogen; periodontal, lytic, or metastatic bone Miacalcic;Miacalcin; disease; osteoclast differentiation SB205614; Macritonin;inhibition; bone disorders; bone Cibacalcin; Cibacalcina; healing andregeneration. Cibacalcine; Salmocalcin; PowderJect Calcitonin)(CAS-21215-62-3) Interferon beta Modulates MHC Anti-viral assay:Multiple Sclerosis; Oncology; (Interferon beta-1a; antigen expression,NK Rubinstein S, Familletti Cancer; Solid Tumors; Melanoma; Interferonbeta 1b; cell activity and IFNg P C, Pestka S. (1981) MalignantMelanoma; Renal Cancer Interferon- beta-serine; production and IL12Convenient assay for (e.g., Renal Cell Carcinoma); Lung SH 579; ZK157046; production in interferons. J. Virol. Cancer (e.g,. Non-SmallCell Lung BCDF; beta-2 IF; monocytes. 37(2): 755-8; Anti- Cancer orSmall Cell Lung Cancer) Interferon- beta-2; rhIL- proliferation assay:Gao Colon Cancer; Breast Cancer; Liver 6; SJ0031; DL 8234; Y, et al(1999) Sensitivity Cancer; Prostate Cancer; Bladder FERON; IFNbeta; ofan epstein-barr virus- Cancer; Gastric Cancer; Sarcoma; BETASERON;positive tumor line, AIDS-Related Kaposi's Sarcoma; AVONEX; REBIF;Daudi, to alpha interferon Lymphoma; T Cell Lymphoma; BETAFERON;correlates with Cutaneous T-Cell Lymphoma; Non- SIGOSIX) expression of aGC-rich Hodgkin's Lymphoma; Brain Cancer; viral transcript. Mol CellGlioma; Glioblastoma Multiforme; Biol. 19(11): 7305-13. CervicalDysplasia; Leukemia; Preleukemia; Bone Marrow Disorders; Bone Disorders;Hairy Cell Leukemia; Chronic Myelogeonus Leukemia; HematologicalMalignancies; Hematological Disorders; Multiple Myeloma; BacterialInfections; Chemoprotection; Thrombocytopenia; Viral infections; HIVInfections; Hepatitis; Chronic Hepatitis; Hepatitis B; Chronic HepatitisB; Hepatitis C; Chronic Hepatitis C; Hepatitis D; Chronic Hepatitis D;Human Papillomavirus; Herpes Simplex Virus Infection; ExternalCondylomata Acuminata; HIV; HIV infection; Pulmonary Fibrosis;Age-Related Macular Degeneration; Macular Degeneration; Crohn's Disease;Neurological Disorders; Arthritis; Rheumatoid Arthritis; UlcerativeColitis; Osteoporosis, Osteopenia, Osteoclastogenesis; Fibromyalgia;Sjogren's Syndrome; Chronic Fatigue Syndrome; Fever; Hemmorhagic Fever;Viral Hemmorhagic Fevers; Hyperglycemia; Diabetes; Diabetes Insipidus;Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non- insulindependent Diabetes Mellitus (NIDDM); Insulin- dependent DiabetesMellitus (IDDM); A Condition Associated With Diabetes Including, But NotLimited To Obesity, Heart Disease, Hyperglycemia, Infections,Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;Obesity; Vascular Disorders; Suppression of Body Weight; Suppression ofAppetite; Syndrome X. Growth hormone Acts on the anterior Growthhormone- Acromegaly; Growth failure; Growth releasing factor; Growthpituitary to stimulate releasing peptides hormone replacement; Growthhormone releasing the production and (GHRPs) are known to hormonedeficiency; Pediatric hormone (Sermorelin secretion of growth releasegrowth hormone Growth Hormone Deficiency; Adult acetate; Pralmorelin;hormone and exert a (GH) in vivo and in vitro Growth Hormone Deficiency;Somatorelin; trophic effect on the by a direct action on IdiopathicGrowth Hormone Somatoliberin; Geref; gland. receptors in anteriorDeficiency; Growth retardation; Gerel; Groliberin) pituitary cells.Biological Prader- Willi Syndrome; Prader-Willi activity can be measuredSyndrome in children 2 years or in cell lines expressing older; Growthdeficiencies; Growth growth hormone failure associated with chronicrenal releasing factor receptor insufficiency; Osteoporosis; (MolEndocrinol 1992 Osteopenia, Osteoclastogenesis; October; 6(10):Postmenopausal osteoporosis; burns; 1734-44, Cachexia; Cancer Cachexia;Molecular Dwarfism; Metabolic Disorders; Endocrinology, Vol 7, Obesity;Renal failure; Turner's 77-84). Syndrome; Fibromyalgia; Fracturetreatment; Frailty, AIDS wasting; Muscle Wasting; Short Stature;Diagnostic Agents; Female Infertility; lipodystrophy. IL-2 (Aldesleukin;Promotes the growth of T cell proliferation assay Cancer; Solid Tumors;Metastatic interleukin-2 fusion B and T cells and “Biological activityof Renal Cell Carcinoma; Metastatic toxin; T cell growth augments NKcell and recombinant human Melanoma; Malignant Melanoma; factor;PROLEUKIN; CTL cell killing interleukin-2 produced in Melanoma; RenalCell Carcinoma; IMMUNACE; activity. Escherichia coli.” Renal Cancer;Lung Cancer (e.g,. CELEUK; ONCOLIPIN Science 223: 1412-1415, Non-SmallCell Lung Cancer or 2; MACROLIN) 1984. natural killer (NK) Small CellLung Cancer); Colon cell and CTL cytotoxicity Cancer; Breast Cancer;Liver Cancer; assay “Control of Leukemia; Preleukemia; homeostasis ofCD8+ Hematological Malignancies; memory T cells by HematologicalDisorders; Acute opposing cytokines. Myeloid Leukemia; Melanoma; Science288: 675-678, Malignant Melanoma; Non- 2000; CTLL-2 Hodgkin's Lymphoma;Ovarian Proliferation: Gillis et al Cancer; Prostate Cancer; Brain(1978) J. Immunol. 120, Cancer; Glioma; Glioblastoma 2027 Multiforme;Hepatitis; Hepatitis C; Lymphoma; HIV Infection (AIDS); InflammatoryBowel Disorders; Kaposi's Sarcoma; Multiple Sclerosis; Arthritis;Rheumatoid Arthritis; Transplant Rejection; Diabetes; Type 1 DiabetesMellitus; Type 2 Diabetes. Parathyroid hormone; Acts in conjuction withAdenylyl cyclase Bone Disorders; Fracture prevention; parathyrin (PTH;calcitonin to control stimulation in rat Hypercalcemia; MalignantOstabolin; ALX1-11; calcium and phosphate osteosarcoma cells,hypercalcemia; Osteoporosis; Paget's hPTH 1-34; LY 333334; metabolism;elevates ovariectomized rat disease; Osteopenia, MN 10T; parathyroidblood calcium level; model of Osteoclastogenesis; osteolysis; hormone(1-31); stimulates the activity osteoporosis: IUBMB osteomyelitis;osteonecrosis; FORTEO; of osteocytes; enhances Life 2000 Februray;periodontal bone loss; osteoarthritis; PARATHAR) absorption of Ca+/Pi49(2): 131-5 rheumatoid arthritis; osteopetrosis; from small intestineperiodontal, lytic, or metastatic bone into blood; promotes disease;osteoclast differentiation reabsorption of Ca+ and inhibition; bonedisorders; bone inhibits Pi by kidney healing and regeneration. tubules.Resistin Mediates insulin Ability of resistin to Hyperglycemia;Diabetes; Diabetes resistance in Type II influence type II diabetesInsipidus; Diabetes mellitus; Type 1 diabetes; inhibits can bedetermined using diabetes; Type 2 diabetes; Insulin insulin-stimulatedassays known in the art: resistance; Insulin deficiency; glucose uptakePontoglio et al., J Clin Hyperlipidemia; Hyperketonemia; Invest 1998 May15; Non- insulin dependent Diabetes 101(10): 2215-22. Mellitus (NIDDM);Insulin- dependent Diabetes Mellitus (IDDM); A Condition Associated WithDiabetes Including, But Not Limited To Obesity, Heart Disease,Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; MetabolicDisorders; Immune Disorders; Obesity; Vascular Disorders; Suppression ofBody Weight; Suppression of Appetite; Syndrome X. TR6 (DcR3; DecoyInhibits Fas Ligand and Cellular apoptosis can Fas Ligand or LIGHTinduced Receptor 3; FASTR) AIM-2 (TL5, LIGHT) be measured by annexinapoptotic disorders; hepatitis; liver mediated apoptosis. staining,TUNEL failure (including fulminant liver staining, measurement offailure); graft versus host disease; caspase levels. Inhibition graftrejection; myelodysplastic of cell growth can also be syndrome; renalfailure; insulin directly measured, for dependent diabetes mellitus;example by ALOMAR rheumatoid arthritis; inflammatory Blue staining.Assay refs: bowel disease; autoimmune disease; cytotoxicity assay ontoxic epidermal necrolysis; multiple human fibrosarcoma sclerosis.(Epsevik and Nissen- Meyer, 1986, J. Immunol. methods). DeCAF (D- SLAM;Inhibits proliferation DeCAF activity can be B cell and/or T cellmediated immune BCM-like membrane and differentiation of B determinedusing assays disorders; Immunodeficiency (e.g., protein; BLAME (B cells;Antagonize BLyS known in the art, such as Common Variable lymphocyteactivator activity for example, those Immunodeficiency, Selective IgAmacrophage described in Examples Deficiency) expressed))x 32-33 ofInternational Publication No. WO0111046. BLyS (B Lymphocyte Promotesproliferation, BLyS activity can be B cell and/or T cell mediated immuneStimulator; Neutrokine differentiation and determined using assaysdisorders, particularly immune alpha; TL7; BAFF; survival of B cells;known in the art, such as, system disorders associated with low TALL-1;THANK; Promotes for example, the B cell numbers or low serumradiolabeled BLyS) immunoglobulin costimulatory immunoglobulin;Immunodeficiency production by B cells. proliferation assay and (e.g.,Common Variable other assays disclosed by Immunodeficiency, SelectiveIgA Moore et al., 1999, Deficiency). Radiolabeled forms; Science,285(5425): 260-3. lymphoma, non-Hodgkins lymphoma, chronic lymphocyticleukemia, multiple myeloma. Anti-BLyS single chain Agonize or antagonizeBLyS agonist or B cell and/or T cell mediated immune antibody(scFvI116A01, BlyS activity. antagonist activity can be disorders;Autoimmune disorders, scFvI050B11, determined using assays particularlyautoimmune diseases scFvI006D08) and known in the art, such as,associated with the production of others. for example, a modifiedautoantibodies; Rheumatoid Arthritis, version the costimulatory SystemicLupus Erythmatosus; proliferation assay Sjogren's Syndrome, cancersdisclosed by Moore et al., expressing Blys as an autocrine 1999,Science, growth factor, e.g. certain chronic 285(5425): 260-3, inlymphocytic leukemias. which BlyS is mixed or preincubated with theanti-BlyS antibody prior to being applied to the responder Blymphocytes. MPIF-1 (Myeloid Inhibits myeloid MPIF-1 activity can beChemoprotection; Adjunct to Progenitor Inhibitory progenitor cells; andmeasured using the Chemotherapy; Inflammatory Factor; CK beta-8;activates monocytes myeloprotection assay disorders; Cancer; Leukemia;Mirostipen) and chemotaxis assay Myelocytic leukemia; Neutropenia,described in U.S. Pat. No. Primary neutropenias (e.g.; 6,001,606.Kostmann syndrome); Secondary neutropenia; Prevention of neutropenia;Prevention and treatment of neutropenia in HIV- infected patients;Prevention and treatment of neutropenia associated with chemotherapy;Infections associated with neutropenias; Myelopysplasia; Autoimmunedisorders; Psoriasis; Mobilization of hematopoietic progenitor cells;Wound Healing; Autoimmune Disease; Transplants; Bone marrow transplants;Acute myelogeneous leukemia; Lymphoma, Non- Hodgkin's lymphoma; Acutelymphoblastic leukemia; Hodgkin's disease; Accelerated myeloid recovery;Glycogen storage disease. KDI (Keratinocyte Inhibits bone marrow KDIactivity can be Multiple sclerosis; Hepatitis; Cancer; DerivedInterferon; proliferation; and shows measured using the Viralinfections, HIV infections, Interferon Kappa antiviral activity.antiviral and cell Leukemia. Precursor) proliferation assays describedin Examples 57-63 of International Publication No. WO0107608. TNFR2(p75) Binds both TNFα and T-cell proliferation can Autoimmune disease;Rheumatoid (ENBREL) TNFβ; mediates T-cell be measured using assaysArthritis; Psoriatic arthritis; Still's proliferation by TNF; known inthe art. For Disease; Ankylosing Spondylitis; reduces signs and example,“Lymphocytes: Cardiovascular Diseases; Vasulitis; structural damage in apractical approach” Wegener's granulomatosis; patients with edited by: SL Rowland, Amyloidosis; Systemic Lupus moderately to severely A JMcMichael - chapter Erythematosus, Insulin-Dependent active rheumatoid6, pages 138-160 Oxford Diabetes Mellitus; Immunodeficiency arthritis(RA). University Press (2000); Disorders; Infection; Inflammation; and“Current Protocols Inflammatory Bowel Disease; on CD-ROM” sectionChrohn's Disease; Psoriasis; AIDS; 3.12 Proliferation Assays GraftRejection; Graft Versus Host for T-cell Function John Disease. Wiley &Soncs, Inc. (1999). Keratinocyte growth Stimulates epithelial KGF-2activity can be Stimulate Epithelial Cell factor 2 (Repifermin; cellgrowth. measured using the Proliferation; Stimulate Basal KGF-2;Fibroblast wound healing assays and Keratinocytes; Wound Healing; GrowthFactor-10; FGF-10) epithelial cell Stimulate Hair Follicle Production;proliferation assays Healing Of Dermal Wounds. Wound described in U.S.Pat. No. Healing; Eye Tissue Wounds, Dental 6,077,692. Tissue Wounds,Oral Cavity Wounds, Diabetic Ulcers, Dermal Ulcers, Cubitus Ulcers,Arterial Ulcers, Venous Stasis Ulcers, Burns Resulting From HeatExposure Or Chemicals, or Other Abnormal Wound Healing Conditions suchas Uremia, Malnutrition, Vitamin Deficiencies or ComplicationsAssociated With Systemic Treatment With Steroids, Radiation Therapy orAntineoplastic Drugs or Antimetabolites; Promote Dermal ReestablishmentSubsequent To Dermal Loss; Increase the Adherence Of Skin Grafts To AWound Bed; Stimulate Re-Epithelialization from The Wound Bed; To PromoteSkin Strength; Improve The Appearance Of Aged Skin; ProliferateHepatocytes, Lung, Breast, Pancreas, Stomach, Bladder, Small Intestine,Large Intestine; Sebocytes, Hair Follicles, Type II Pneumocytes, Mucin-Producing Goblet Cells, or Other Epithelial Cells, Endothelial Cells,Keratinocytes, or Basal Keratinocytes (and Their Progenitors) ContainedWithin The Skin, Lung, Liver, Bladder, Eye, Salivary Glands, orGastrointestinal Tract; Reduce The Side Effects Of Gut Toxicity ThatResult From Radiation, Chemotherapy Treatments Or Viral Infections;Cytoprotector, especially of the Small Intestine Mucosa or Bladder;Mucositis (Mouth Ulcers); Regeneration Of Skin; Full and/or PartialThickness Skin Defects, including Burns, (e.g., Repopulation Of HairFollicles, Sweat Glands, And Sebaceous Glands); Psoriasis; EpidermolysisBullosa; Blisters; Gastric and/or Doudenal Ulcers; Reduce Scarring;Inflamamatory Bowel Diseases; Crohn's Disease; Ulcerative Colitis; GutToxicity; Lung Damage; Repair Of Alveoli And/or Brochiolar Epithelium;Acute Or Chronic Lung Damage; Emphysema, ARDS; Inhalation Injuries;Hyaline Membrane Diseases; Infant Respiratory Distress Syndrome;Bronchopulmonary Displasia In Premature Infants; Fulminant LiverFailure; Cirrhosis, Liver Damage caused by Viral Hepatitis and/or ToxicSubstances; Diabetes Mellitus; Inflammation. TR2 (and TR2sv1, Inhibits Bcell Co-stimulation B-cell Herpes; immune disorders; TR2SV2; TNFRSF14;proliferation, and proliferation assay and Ig autoimmune disease; graftversus HVEM; Herpes Virus mediates and inhibits production assay (Moorehost disease; graft rejection; variable Entry Mediator; ATAR) HerpesSimplex Virus et al., 1999, Science, immunodeficiency; (HSV) infection.285(5425): 260-3.). immunodeficiency syndromes; HSV-1 and HSV-2 cancer.Infectivity Assay: International Publication No. WO 97/ Macrophagederived Chemotactic for 04658 Chemokine Inflammatory diseases; woundchemokine, MDC monocyte-derived activities can be healing; (Ckbeta-13)dendritic cells and IL-2- determined using assays angiogenesis; AIDSinfection. activated natural killer known in the art: cells. Methods inMolecular Biology, 2000, vol. 138: Chemokine Protocols. Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power.  ©Humana Press Inc.,Totowa, NJ HAGDG59 Activates MIP1a Dendritic cell assays Immunedisorders; cancer; viral (Retinal release in Dendritic are well known inthe infection; inflammation; sepsis; short-chain Cells. art. Forexample, J. arthritis; asthma. dehydrogenase) Immunol. 158: 2919-2925(1997); J. Leukoc. Biol. 65: 822-828 (1999). GnRH (Gonadotropin Promotesrelease of GnRH is known to cause Infertility; Kallmann's syndrome orReleasing Hormone) follicle-stimulating the release of follicle otherforms of hypergonadotropic hormone and stimulating hormone hypergonadism(failure to go through luteinizing hormone (FSH) and/or luteinizingpuberty naturally). from anterior pituitary. hormone (LH) in vivo by adirect action on receptors in anterior pituitary gonadotropes. GnRHactivity can be determined by measuring FSH levels in the medium ofcultured gonadotropes before and after GnRH supplementation. Forexample, Baker et al. Biol Reprod 2000 September; 63(3): 865-71.Teprotide Inhibits angiotensin Inhibition of ACE can be Hypertension;congestive heart converting enzyme determined using assays failure.(ACE). known in the art. For example, Anzenbacherova et al., J. PharmaBiomed Anal 2001 March; 24(5-6): 1151-6. Human chemokine Involved inChemokine activities can Autoimmune disorders; Immunity; HCC-1(ckBeta-1; inflammation, allergy, be determined using Vascular andInflammatory disorders; HWFBD) tissue rejection, viral assays known inthe art: HIV; AIDS; infectious diseases. infection, and tumor Methods inMolecular biology; enhances Biology, 2000, vol. 138: proliferation ofCD34+ Chemokine Protocols. myeloid progenitor Edited by: A. E. I. cells.Proudfoot, T. N. C. Wells, and C. A. Power.  ©Humana Press Inc., Totowa,NJ ACE2 inhibitor Inhibits production of Inhibition of angiotensinTreatment for elevated angiotensin II (DX512) angiotensin II which canbe determined using and/or aldosterone levels, which can inducesaldosterone assays known in the art. lead to vasoconstriction, impairedproduction, arteriolar For example, in vitro cardiac output and/orhypertension; smooth muscle using a proliferation CardiovascularDisease; Cardiac vasoconstriction, and assay with rat cardiac Failure;Diabetes; Type II Diabetes; proliferation of cardiac fibroblasts asdescribed in Proteinuria; Renal disorders, fibroblasts, Induces NaunynSchmiedebergs congestive heart failure. angiogenesis; an Arch Pharmacol1999 enzyme that converts May; 359(5): 394-9. angiotensin I toangiotensin1-9; also cleaves des-Arg, bradykinin and neurotensin. TR1(OCIF; Inhibits Coculture Assay for Osteoporosis; Paget's disease;Osteoclastogenesis osteoclastogenesis and Osteoclastogenesis, Boneosteopenia; osteolysis; osteomyelitis; inhibitory factor; boneresorption, and resorption assay using osteonecrosis; periodontal boneloss; osteoprotegerin, OPG; induces fibroblast fetal long-bone organosteoarthritis; rheumatoid arthritis; tumor necrosis factorproliferation. culture system, dentine osteopetrosis; periodontal,lytic, or receptor superfamily resorption assay, and metastatic bonedisease; osteoclast member 11B precursor;) fibroblast proliferationdifferentiation inhibition; bone assays are each described disorders;bone healing and in Kwon et al., FASEB J. regeneration; organcalcification; 12: 845-854 (1998). vascular calcification. Humanchemokine Chemotactic for both Chemokine activities can Cancer; Woundhealing; Ckbeta-7 activated (CD3+) T be determined using Inflammatorydisorders; cells and nonactivated assays known in the art:Immmunoregulatory disorders; (CD14−) lymphocytes Methods in MolecularAtherosclerosis; Parasitic Infection; and (CD4+) and Biology, 2000, vol.138: Rheumatoid Arthritis; Asthma; (CD8+) T lymphocytes ChemokineProtocols. Autoimmune disorders. and (CD45RA+) T cells Edited by: A. E.I. Proudfoot, T. N. C. Wells, and C. A. Power.  ©Humana Press Inc.,Totowa, NJ CKbeta4 (HGBAN46; Attracts and activates Chemokine activitiescan Cancer; Solid Tumors; Chronic HE9DR66) microbicidal be determinedusing Infection; Autoimmune Disorders; leukocytes; Attracts assays knownin the art: Psoriasis; Asthma; Allergy; CCR6-expressing Methods inMolecular Hematopoiesis; Wound Healing; immature dendritic cellsBiology, 2000, vol. 138: Bone Marrow Failure; Silicosis; andmemory/effector T Chemokine Protocols. Sarcoidosis; Hyper-Eosinophiliccells; B-cell Edited by: A. E. I. Syndrome; Lung Inflammation;chemotaxis; inhibits Proudfoot, T. N. C. Fibrotic Disorders;Atherosclerosis; proliferation of myeloid Wells, and C. A. Power.Periodontal diseases; Viral diseases; progenitors; chemotaxis  ©HumanaPress Inc., Hepatitis. of PBMC's. Totowa, NJ Leptin Controls obesity invivo modulation of Hyperglycemia; Diabetes; Diabetes through regulationof food intake, reduction in Insipidus; Diabetes mellitus; Type 1appetite, reduction of body weight, and diabetes; Type 2 diabetes;Insulin body weight, and lowering of insulin and resistance; Insulindeficiency; lowering of insulin and glucose levels in ob/obHyperlipidemia; Hyperketonemia; glucose level. mice, radioimmunoassayNon-insulin dependent Diabetes (RIA) and activation of Mellitus (NIDDM);Insulin- the leptin receptor in a dependent Diabetes Mellitus cell-basedassay. Protein (IDDM); a Condition Associated Expr Purif 1998 December;With Diabetes Including, But Not 14(3): 335-42 Limited To Obesity, HeartDisease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers;Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;Suppression of Body Weight; Suppression of Appetite; Syndrome X;Immunological Disorders; Immunosuppression. IL-1 receptor antagonistBinds IL1 receptor 1) Competition for IL-1 Autoimmune Disease;Arthritis; (Anakinra; soluble without activating the binding to IL-1receptors Rheumatoid Arthritis; Asthma; interleukin-1 receptor; targetcells; inhibits the in YT-NCI or C3H/HeJ Diabetes; Diabetes Mellitus;GVHD; IRAP; KINERET; binding of IL1-alpha cells (Carter et al., NatureInflammatory Bowel Disorders; ANTRIL) and IL1-beta; and 344: 633-638,1990); 2) Chron's Disease; Ocular neutralizes the biologic Inhibition ofIL-1- Inflammation; Psoriasis; Septic activity of IL1-alpha inducedendothelial cell- Shock; Transplant Rejection; and IL1-beta. leukocyteadhesion Inflammatory Disorders; Rheumatic (Carter et al., Nature 344:Disorders; Osteoporosis; 633-638, 1990); 3) Postmenopausal Osteoporosis;Proliferation assays on Stroke. A375-C6 cells, a human melanoma cellline highly susceptible to the antiproliferative action of IL-1 (Murai Tet al., J. Biol. Chem. 276: 6797- 6806, 2001). TREM-1 (TriggeringMediates activation of Secretion of cytokines, Inflammation; Sepsis;bacterial Receptor Expressed on neutrophil and chemokines, infection;autoimmune diseases; Monocytes 1) monocytes; Stimulates degranulation,and cell GVHD. neutrophil and surface activation monocyte-mediatedmarkers can be inflammatory response; determined using assays Promotessecretion of described in Bouchon et TNF, IL-8, and MCP-1; al, J Immunol2000 May Induces neutrophil 15; 164(10): 4991-5. degranulation, Ca2+mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C- gamma. HCNCA73Induces T-cell FMAT can be used to Autoimmune disorders; Inflammationactivation- expression measure T-cell surface of the gastrointestinaltract; Cancer; of CD152 marker; markers (CD69, CD152, Colon Cancer;Allergy; Crohn's Stimulates release of CD71, HLA-DR) and T- disease.TNF-a and MIP- 1a cell cytokine production from immature, (e.g., IFNgproduction). monocyte-derived J. of Biomol. Screen. 4: dendritic cells;193-204 (1999). Other T- Promotes maturation of cell proliferationassays: dendritic cells. “Lymphocytes: a practical approach” edited by:S L Rowland, A J McMichael - Chapter 6, pages 138-160 Oxford UniversityPress (2000); WO 01/21658 Examples 11-14, 16-17 and 33. VEGF-2 (VascularPromotes endothelial VEGF activity can be Coronary artery disease;Critical limb Endothelial Growth cell proliferation. determined usingassays ischemia; Vascular disease; Factor-2; VEGF-C) known in the art,such as proliferation of endothelial cells, both those disclosed invascular and lymphatic. Antagonists International Publication may beuseful as anti-angiogenic No. WO0045835, for agents; Cancer. example.HCHNF25 (jumping Activates MIP1a Dendritic cell assays are Immunedisorders; cancer. translocation Release in Dendritic well known in theart. For breakpoint) Cells. example, J. Immunol. 158: 2919-2925 (1997);J. Leukoc. Biol. 65: 822- 828 (1999). HLDOU18 (Bone Activates L6/GSK3Assays for activation of Hyperglycemia; Diabetes; Diabetes MorphogenicProtein 9 kinase assay. GSK3 kinase activity are Insipidus; Diabetesmellitus; Type 1 (BMP9); Growth well known in the art. For diabetes;Type 2 diabetes; Insulin differentiation factor-2 example, Biol. Chem.resistance; Insulin deficiency; precursor (GDF-2 379(8-9): (1998) 1101-Hyperlipidemia; Hyperketonemia; precursor)) 1110.; Biochem J. 1993 Non-insulin dependent Diabetes Nov. 15; 296 (Pt 1): 15-9. Mellitus (NIDDM);Insulin- dependent Diabetes Mellitus (IDDM); A Condition Associated WithDiabetes Including, But Not Limited To Obesity, Heart Disease,Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; MetabolicDisorders; Immune Disorders; Obesity; Vascular Disorders; Suppression ofBody Weight; Suppression of Appetite; Syndrome X. Glucagon- Like-PeptideStimulates the synthesis GLP1 activity may be Hyperglycemia; Diabetes;Diabetes 1 (GLP1; and release of insulin; assayed in vitro using aInsipidus; Diabetes mellitus; Type 1 Insulinotropin) enhances thesensitivity [3-H]-glucose uptake diabetes; Type 2 diabetes; Insulin ofadipose, muscle, and assay. (J Biol Chem 1999 resistance; Insulindeficiency; liver tissues towards Oct. 22; 274(43): 30864-Hyperlipidemia; Hyperketonemia; insulin; stimulates 30873). Non- insulindependent Diabetes glucose uptake; slows Mellitus (NIDDM); Insulin- thedigestive process; dependent Diabetes Mellitus suppresses appetite;(IDDM); A Condition Associated blocks the secretion of With DiabetesIncluding, But Not glucagon. Limited To Obesity, Heart Disease,Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; MetabolicDisorders; Immune Disorders; Obesity; Vascular Disorders; Suppression ofBody Weight; Suppression of Appetite; Syndrome X. Exendin-4 (AC-2993)Stimulates the synthesis Exendin-4 activity may Hyperglycemia; Diabetes;Diabetes and release of insulin; be assayed in vitro using Insipidus;Diabetes mellitus; Type 1 enhances the sensitivity a [3-H]-glucoseuptake diabetes; Type 2 diabetes; Insulin of adipose, muscle, and assay.(J Biol Chem 1999 resistance; Insulin deficiency; liver tissues towardsOct. 22; 274(43): Hyperlipidemia; Hyperketonemia; insulin; stimulates30864-30873). Non- insulin dependent Diabetes glucose uptake; slowsMellitus (NIDDM); Insulin- the digestive process; dependent DiabetesMellitus suppresses appetite; (IDDM); A Condition Associated blocks thesecretion of With Diabetes Including, But Not glucagon. Limited ToObesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/OrUlcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. T20 (T20 HIV a peptide from residues Virus inhibition assays as HIV;AIDS; SIV (simian inhibitory peptide, 643-678 of the HIV described inZhang et al., immunodeficiency virus) infection. DP178; DP178 HIV gp41transmembrane Sep. 26, 2002, inhibitory peptide) protein ectodomainSciencexpress which binds to gp41 in (www.sciencexpress.org). itsresting state and prevents transformation to the fusogenic state T1249(T1249 HIV a second generation Virus inhibition assays as HIV; AIDS; SIV(simian inhibitory peptide; HIV fusion inbitor described in Zhang etal., immunodeficiency virus) infection T1249 anti-HIV Sep. 26, 2002,peptide) Sciencexpress (www.sciencexpress.org). Interferon Hybrids,Confers a range of Anti-viral assay: Viral infections; HIV Infections;specifically preferred: cellular responses Rubinstein S, FamillettiHepatitis; Chronic Hepatitis; IFNalpha A/D hybrid including antiviral PC, Peslka S. (1981) Hepatitis B; Chronic Hepatitis B; (BgIII version)antiproliferative, Convenient assay for Hepatitis C; Chronic HepatitisC; IFNalpha A/D hybrid antitumor and interferons. J. Virol. Hepatitis D;Chronic Hepatitis D; (PvuII version) immunomodulatory 37(2): 755-8;Anti- Human Papillomavirus; Herpes IFNalpha A/F hybrid activities;stimulate proliferation assay: Gao Simplex Virus Infection; ExternalIFNalpha A/B hybrid production of two Y, et al (1999) SensitivityCondylomata Acuminata; HIV; HIV IFNbeta 1/alpha D enzymes: a protein ofan epstein-barr virus- Infection; Oncology; Cancer; Solid hybrid(IFNbeta- kinase and an positive tumor line. Tumors; Melanoma; Malignant1/alpha-1 hybrid) oligoadenylate Daudi, to alpha interferon Melanoma;Renal Cancer (e.g., Renal IFNalpha/beta hybrid synthetase. Also,correlates with Cell Carcinoma); Lung Cancer (e.g., modulates MHCexpression of a GC-rich Non-Small Cell Lung Cancer or antigenexpression, NK viral transcript. Mol Cell Small Cell Lung Cancer) Coloncell activity and IFNg Biol. 19(11): 7305-13. Cancer; Breast Cancer;Liver Cancer; production and IL12 Prostate Cancer; Bladder Cancer;production in Gastric Cancer; Sarcoma; AIDS- monocytes. Related Kaposi'sSarcoma; Lymphoma; T Cell Lymphoma; Cutaneous T-Cell Lymphoma; Non-Hodgkin's Lymphoma; Brain Cancer; Glioma; Glioblastoma Multiforme;Cervical Dysplasia; Leukemia; Preleukemia; Bone Marrow Disorders; BoneDisorders; Hairy Cell Leukemia; Chronic Myelogeonus Leukemia;Hematological Malignancies; Hematological Disorders; Multiple Myeloma;Bacterial Infections; Chemoprotection; Thrombocytopenia; MultipleSclerosis; Pulmonary Fibrosis; Age- Related Macular Degeneration;Macular Degeneration; Crohn's Disease; Neurological Disorders;Arthritis; Rheumatoid Arthritis; Ulcerative Colitis; Osteoporosis,Osteopenia, Osteoclastogenesis; Fibromyalgia; Sjogren's Syndrome;Chronic Fatigue Syndrome; Fever; Hemmorhagic Fever; Viral HemmorhagicFevers; Hyperglycemia; Diabetes; Diabetes Insipidus; Diabetes mellitus;Type 1 diabetes; Type 2 diabetes; insulin resistance; Insulindeficiency; Hyperlipidemia; Hyperketonemia; Non- insulin dependentDiabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus (IDDM);A Condition Associated With Diabetes Including, But Not Limited ToObesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/OrUlcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. B-type natriuretic stimulates smooth Inhibition of angiotensinCongestive heart failure; cardiac peptide (BNP, brain muscle relaxationand can be determined using volume overload; cardiac natriureticpeptide) vasodilation, assays known in the art, decompensation; CardiacFailure; natriuresis, and for example using an in Left VentricularDysfunction; suppression of renin- vitro proliferation assay Dyspneaangiotensin and with rat cardiac endothelin. fibroblasts as described inNaunyn Schmiedebergs Arch Pharmacol 1999 May; 359(5): 394-9.Vasodilation can be measured in animals by measuring the myogenicresponses of small renal arteries in an isobaric arteriograph system(see Am J Physiol Regul Integr Comp Physiol 2002 August; 283(2): R349-R355). Natriuesis is determined by measuring the amount of sodium in theurine. α-defensin, including Suppression of HIV Virus inhibition assaysas HIV, AIDS; ARC. alpha 1 defensin, alpha 2 replication; activedescribed in Zhang et al., defensin, alpha 3 against bacteria, fungi,Sep. 26, 2002, defensin (myeloid- and enveloped viruses. Sciencexpressrelated defensin; (www.sciencexpress.org). DEFA1; neutrophil- specificdefensin; CAF) Phosphatonin (matrix Regulation of Blood phosphate levelsHyperphosphatemia; extracellular phosphate metabolism. can be measuredusing Hyperphosphatemia in chronic renal phosphoglycoprotein; methodsknown in the art failure; hypophosphatemia; MEPE) such as theOsteomalacia; Rickets; X-linked Hypophosphatemic Rat dominanthypophosphatemic Bioassay. Zoolog Sci rickets/osteomalacia (XLH) ; 1995October; 12(5): autosomal dominant 607-10, hypophosphatemicrickets/osteomalacia (ADHR); tumor- induced rickets/osteomalacia (TIO).P1pal-12 (pepducin, Regulation of protease- Platelet aggregation canProtection against systemic platelet PAR1-based pepducin) activatedreceptor be measured using activation, thrombus, heart attack, (PAR)signal methods known in the art stroke, and/or coagulation disorders.transduction and such as described in thrombin-mediated Nature Medicine2002 aggregation of human October; 8(10): 1161-1165. platelets. P4pal-10(pepducin, Regulation of protease- Platelet aggregation can Protectionagainst systemic platelet PAR4-based pepducin) activated receptor bemeasured using activation, thrombus, heart attack, (PAR) signal methodsknown in the art stroke, and/or coagulation disorders. transduction andsuch as described in thrombin-mediated Nature Medicine 2002 aggregationof human October; 8(10): 1161-1165. platelets. HRDFD27 Involved in theT-cell proliferation can Chemoprotection; Adjunct to proliferation of Tcells; be measured using assays Chemotherapy; Inflammatory Production ofknown in the art. For disorders; Cancer; Leukemia; TNFgamma. example,“Lymphocytes: Myelocytic leukemia; Neutropenia, a practical approach”Primary neutropenias (e.g.; edited by: S L Rowland, Kostmann syndrome);Secondary A J McMichael - chapter neutropenia; Prevention of 6, pages138-160 Oxford neutropenia; Prevention and University Press (2000);treatment of neutropenia in HIV- and “Current Protocols infectedpatients; Prevention and on CD-ROM” section treatment of neutropeniaassociated 3.12 Proliferation Assays with chemotherapy; Infections forT-cell Function John associated with neutropenias; Wiley & Soncs, Inc.Myelopysplasia; Autoimmune (1999). disorders; Psoriasis; Mobilization ofhematopoietic progenitor cells; Wound Healing; Autoimmune Disease;Transplants; Bone marrow transplants; Acute myelogeneous leukemia;Lymphoma, Non- Hodgkin's lymphoma; Acute lymphoblastic leukemia;Hodgkin's disease; Accelerated myeloid recovery; Glycogen storagedisease HWHGZ51 (CD59; Stimulates an immune The ability to affectSkeletal diseases and disorders; Metastasis- associated response andinduces chondrocyte Musculoskeletal diseases and GPI-adhered proteininflammation by differentiation can be disorders; Bone fractures and/orhomolog) inducing mononuclear measured using methods breaks;Osteoporosis cell, eosinophil and known in the art, such as(postmenopausal, senile, or idiopathic PMN infiltration; described inBone (1995) juvenile); Gout and/or pseudogout; Inhibits growth ofSeptember; 17(3): Paget's disease; Osteoarthritis; breast cancer,ovarian 279-86. Tumors and/or cancers of the bone cancer, leukemia, and(osteochondromas, benign melanoma; chondromas, chondroblastomas,Overexpressed in colon, chondromyxoid fibromas, osteoid lung, breast andrectal osteomas, giant cell tumors, multiple tumors; Regulates myelomas,osteosarcomas, glucose and/or FFA fibrosarcomas, malignant fibrousupdate by adipocytes histiocytomas, chondrosarcomas, and skeletalmuscle; Ewing's tumors, and/or malignant Induces lymphomas); Bone andjoint redifferentiation of infections (osteomyelitits and/orchondrocytes infectious arthritis); Charcot's joints; Heel spurs;Sever's disease; Sport's injuries; Cancer; Solid Tumors; Melanoma;Malignant Melanoma; Renal Cancer (e.g., Renal Cell Carcinoma); LungCancer (e.g.. Non- Small Cell Lung Cancer or Small Cell Lung Cancer)Colon Cancer; Breast Cancer; Liver Cancer; Prostate Cancer; BladderCancer; Gastric Cancer; Sarcoma; AIDS-Related Kaposi's Sarcoma;Lymphoma; T Cell Lymphoma; Cutaneous T-Cell Lymphoma; Non-Hodgkin'sLymphoma; Brain Cancer; Glioma; Glioblastoma Multiforme; CervicalDysplasia; Leukemia; Preleukemia; Bone Marrow Disorders; Bone Disorders;Hairy Cell Leukemia; Chronic Myelogeonus Leukemia; HematologicalMalignancies; Hematological Disorders; Multiple Myeloma; Kidney diseasesand disorders; Shonlein-Henoch purpura, Berger disease, celiac disease,dermatitis herpetiformis, Chron disease; Diabetes; Diabetes Insipidus;Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non- insulindependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus(IDDM); A Condition Associated With Diabetes Including, But Not LimitedTo Obesity, Heart Disease, Hyperglycemia, Infections, Retinopathy,And/Or Ulcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX; Kidney disorders; Hyperinsulinemia; Hypoinsulinemia; Immunologicaldisorders (e.g. arthritis, asthma, immunodeficiency diseases, AIDS,rheumatoid arthritis, granulomatous disease, inflammatory bowl disease,sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities,T-cell mediated cytotoxicity, host-versus-graft disease, auto immunitydisorders, demyelination, systemic lupus erythematosis, drug inducedhemolytic anemia, rheumatoid arthritis, Sjorgren's disease, scleroderma)C17 (cytokine- like Inhibits glucose and/or Proliferation of kidneyKidney diseases and disorders; protein C17) FFA uptake by mesangialcells can be Shonlein- Henoch purpura, Berger adipocytes; Inducesassayed using techniques disease, celiac disease, dermatitisproliferation of kidney described in J. Investig. herpetiformis, Chrondisease; mesangial cells; Med. (1998) August; Diabetes; DiabetesInsipidus; Regulation of cytokine 46(6): 297-302, Diabetes mellitus;Type 1 diabetes; production and antigen Type 2 diabetes; Insulinresistance; presentation Insulin deficiency; Hyperlipidemia;Hyperketonemia; Non-insulin dependent Diabetes Mellitus (NIDDM);Insulin- dependent Diabetes Mellitus (IDDM); A Condition Associated WithDiabetes Including, But Not Limited To Obesity, Heart Disease,Hyperglycemia, Infections, Retinopathy, And/Or Ulcers; MetabolicDisorders; Immune Disorders; Obesity; Vascular Disorders; Suppression ofBody Weight; Suppression of Appetite; Syndrome X; Kidney disorders;Hyperinsulinemia; Hypoinsulinemia; Hematopoietic disorders;Immunological diseases and disorders; Developmental diseases anddisorders; Hepatic diseases and disorders; Cancer (particularlyleukemia); Immunological disorders (e.g. arthritis, asthma,immunodeficiency diseases, AIDS, rheumatoid arthritis, granulomatousdisease, inflammatory bowl disease, sepsis, acne, neutropenia,neutrophilia, psoriasis, hypersensitivities, T-cell mediatedcytotoxicity, host- versus-graft disease, autoimmunity disorders,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjorgren's disease, scleroderma) HDPBQ71Regulates production Such assays that may be Blood disorders andinfection (e.g., and secretion of used or routinely viral infections,tuberculosis, IFN gamma; Activation modified to test infectionsassociated with chronic of myeloid cells and/or immunomodulatorygranulomatosus disease and hematopoietic cells activity of polypeptidesmalignant osteoporosis); of the invention Autoimmune disease (e.g.,(including antibodies and rheumatoid arthritis, systemic lupus agonistsor antagonists of erythematosis, multiple sclerosis); the invention)include the Immunodeficiency, boosting a T cell- assays disclosed inmediated immune response, and Miraglia el al., J suppressing a T cell-mediated Biomolecular Screening immune response; Inflammation and 4:193-204 (1999); inflammatory disorders; Idiopathic Rowland et al.,pulmonary fibrosis; Neoplastic ““Lymphocytes: a diseases (e.g.,leukemia, lymphoma, practical approach”” melanoma); Neoplasms andcancers, Chapter 6: 138-160 such as, for example, leukemia, (2000);Gonzalez et al., J lymphoma, melanoma, and prostate, Clin Lab Anal 8(5):225- breast, lung, colon, pancreatic, 233 (1995); Billiau et al.,esophageal, stomach, brain, liver and Ann NY Acad Sci 856: urinarycancer;. Benign 22-32 (1998); Boehm et dysproliferative disorders andpre- al., Annu Rev Immunol neoplastic conditions, such as, for 15:749-795 (1997), and example, hyperplasia, metaplasia, Rheumatology(Oxford) and/or dysplasia; Anemia; 38(3): 214-20 (1999) Pancytopenia;Leukopenia; Thrombocytopenia; Hodgkin's disease; Acute lymphocyticanemia (ALL); Plasmacytomas; Multiple myeloma; Burkitt's lymphoma;Arthritis; AIDS; Granulomatous disease; Inflammatory bowel disease;Sepsis; Neutropenia; Neutrophilia; Psoriasis; Suppression of immunereactions to transplanted organs and tissues; Hemophilia;Hypercoagulation; Diabetes mellitus; Endocarditis; Meningitis; LymeDisease; Asthma; Allergy Oscar (osteoclast- Regulator of osteoclastAssay to detect osteoclast Skeletal diseases and disorders; associatedreceptor differentiation; differentiation is Musculoskeletal diseasesand isoform-3) regulator of innate and described in J. Exp. Med.disorders; Bone fractures and/or adaptive immune (2002) Jan. 21; 195(2):breaks; Osteoporosis responses 201-9. (postmenopausal, senile, oridiopathic juvenile); Gout and/or pseudogout; Paget's disease;Osteoarthritis; Tumors and/or cancers of the bone (osteochondromas,benign chondromas, chondroblastomas, chondromyxoid fibromas, osteoidosteomas, giant cell tumors, multiple myelomas, osteosarcomas,fibrosarcomas, malignant fibrous histiocytomas, chondrosarcomas, Ewing'stumors, and/or malignant lymphomas); Bone and joint infections(osteomyelitits and/or infectious arthritis); Charcot's joints; Heelspurs; Sever's disease; Sport's injuries Tumstatin (T5, T7 or T8Inhibits angiogenesis; A tumor cell proliferation Cancer; Solid Tumors;Melanoma; peptide; α3(IV)NC1) Inhibits tumor growth; assay is describedin J. Malignant Melanoma; Renal Cancer Inhibits protein Biol. Chem.(1997) 272: (e.g., Renal Cell Carcinoma); Lung synthesis 20395-20401,Protein Cancer (e.g,. Non-Small Cell Lung synthesis can be Cancer orSmall Cell Lung Cancer) measured as described in Colon Cancer; BreastCancer; Liver Science (2002) Jan. 4; Cancer; Prostate Cancer; Bladder295(5552): 140-3. Cancer; Gastric Cancer; Sarcoma; AIDS- RelatedKaposi's Sarcoma; Lymphoma; T Cell Lymphoma; Cutaneous T-Cell Lymphoma;Non- Hodgkin's Lymphoma; Brain Cancer; Glioma; Glioblastoma Multiforme;Cervical Dysplasia; Leukemia; Preleukemia; Bone Marrow Disorders; BoneDisorders; Hairy Cell Leukemia; Chronic Myelogeonus Leukemia;Hematological Malignancies; Hematological Disorders; Multiple Myeloma;Angiogenesis CNTF (Ciliary Enhances myelin Regulation of myelinNeurological and neural diseases and neurotrophic factor) formation;Reduces formation can be assayed disorders, particularly diseases andphotoreceptor as described in J. disorders associated with myelin anddegradation; Regulates Neurosci. (2002) Nov. 1; demyelination, such as,for example, calcium currents 22(21): 9221-7. ALS, multiple sclerosis,Huntington's disease; Neuronal and spinal cord injuries; Disorders ofthe eye, such as, for example, retinitis pigmentosa, blindness,color-blindness, macular degeneration. Somatostatin Inhibits growthInhibition of growth Cancer; Metastatic carcinoid tumors; (Octreotide;octreotide hormone, glucagons hormone release in Vasoactive IntestinalPeptide acetate; Sandostating and insulin; Suppresses humans bysomatostatin secreting adenomas; Diarrhea and LAR ®) LF response toGnRH; can be measured as Flushing; Prostatic disorders and Decreasessplanchnic described in J. Clin. cancers; Breast cancer; blood flow;Inhibits Endocrinol. Metab. Gastrointestinal disorders and release ofserotonin, (1973) October; 37(4): cancers; Cancers of the endocrinegastrin, vasoactive 632-4. system; Head and neck intestinal peptide,Inhibition of insulin paragangliomas; Liver disorders and secretin,motilin, and secretion by somatostatin cancers; Nasopharyngeal cancers;pancreatic polypeptide. can be measured as Thyroid disorders andcancers; described in the Lancet Acromegaly; Carcinoid Syndrome; (1973)Dec. 8; 2(7841): Gallbladder disorders, such as 1299-1301. gallbladdercontractility diseases and abnormal bile secretion; Psoriasis; Diabetes;Diabetes Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2 diabetes;Insulin resistance; Insulin deficiency; Hyperlipidemia; Hyperketonemia;Non- insulin dependent Diabetes Mellitus (NIDDM); Insulin-dependentDiabetes Mellitus (IDDM); A Condition Associated With DiabetesIncluding, But Not Limited To Obesity, Heart Disease, Hyperglycemia,Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; ImmuneDisorders; Obesity; Vascular Disorders; Suppression of Body Weight;Suppression of Appetite; Syndrome X; Kidney disorders; Neurologicaldisorders and diseases, including Alzheimers Disease. Parkinson'sdisease and dementia; Neuropsychotic disorders, including Bipolaraffective disorder; Rheumatoid arthritis; Hypertension; Intracranialhypertension; Esophageal varices; Graves' disease; Seizures; Epilepsy;Gastritis; Angiogenesis; IL-22 (IL22, interleukin- Stimulates glucoseIL-22 activity may be Hyperglycemia; Diabetes; Diabetes 22; IL17D, 1L27)uptake in skeletal assayed in vitro using a Insipidus; Diabetesmellitus; Type 1 muscle cells; increases [3-H]-glucose uptake diabetes;Type 2 diabetes; Insulin skeletal muscle insulin assay. (J Biol Chem1999 resistance; Insulin deficiency; sensitivity. Oct. 22; 274(43):30864- Hyperlipidemia; Hyperketonemia; 30873). Non- insulin dependentDiabetes Mellitus (NIDDM); Insulin- dependent Diabetes Mellitus (IDDM);A Condition Associated With Diabetes Including, But Not Limited ToObesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/OrUlcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. HCE1P80 Stimulates glucose HCE1P80 activity may Hyperglycemia;Diabetes; Diabetes uptake in; increases be assayed in vitro usingInsipidus; Diabetes mellitus; Type 1 insulin sensitivity, a[3-H]-glucose uptake diabetes; Type 2 diabetes; Insulin assay. (J BiolChem 1999 resistance; Insulin deficiency; Oct. 22; 274(43): 30864-Hyperlipidemia; Hyperketonemia; 30873), Non- insulin dependent DiabetesMellitus (NIDDM); Insulin- dependent Diabetes Mellitus (IDDM); ACondition Associated With Diabetes Including, But Not Limited ToObesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/OrUlcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. HDRMI82 Stimulates glucose HDRMI82 activity may Hyperglycemia;Diabetes; Diabetes uptake; increases be assayed in vitro usingInsipidus; Diabetes mellitus; Type 1 insulin sensitivity. a[3-H]-glucose uptake diabetes; Type 2 diabetes; Insulin assay. (J BiolChem 1999 resistance; Insulin deficiency; Oct. 22; 274(43): 30864-Hyperlipidemia; Hyperketonemia; 30873), Non- insulin dependent DiabetesMellitus (NIDDM); Insulin- dependent Diabetes Mellitus (IDDM); ACondition Associated With Diabetes Including, But Not Limited ToObesity, Heart Disease, Hyperglycemia, Infections, Retinopathy, And/OrUlcers; Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. HDALV07 Modulates insulin Insulin activity may be Diabetes; DiabetesInsipidus; (adiponectin; gelatin- action assayed in vitro using aDiabetes mellitus; Type 1 diabetes; binding 28k protein [3-H]-glucoseuptake Type 2 diabetes; Insulin resistance; precursor; adipose mostassay. (J Biol Chem 1999 Insulin deficiency; Hyperlipidemia; abundantgene Oct. 22; 274(43): 30864- Hyperketonemia; Non-insulin transcript;APM-1; 30873), dependent Diabetes Mellitus GBP28; ACRP30; (NIDDM);Insulin- dependent ADIPOQ) Diabetes Mellitus (IDDM); A ConditionAssociated With Diabetes Including, But Not Limited To Obesity, HeartDisease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers;Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;Suppression of Body Weight; Suppression of Appetite; Syndrome X;Hyperglycemia; Familial combined hyperlipidemia; Metabolic syndrome;Inflammatory disorders; Atherogenic disorders C Peptide An insulinprecursor C-peptide concentrations Diabetes; Diabetes Insipidus;involved in insulin can be measured using Diabetes mellitus; Type 1diabetes; regulation assays well known in the Type 2 diabetes; Insulinresistance; art, such as the one Insulin deficiency; Hyperlipidemia;described in PNAS Hyperketonemia; Non-insulin (1970) September; 67(1):dependent Diabetes Mellitus 148-55 (NIDDM); Insulin- dependent DiabetesMellitus (IDDM); A Condition Associated With Diabetes Including, But NotLimited To Obesity, Heart Disease, Hyperglycemia, Infections,Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;Obesity; Vascular Disorders; Suppression of Body Weight; Suppression ofAppetite; Syndrome X; Hyperglycemia; Familial combined hyperlipidemia;Metabolic syndrome HCBOG68 (enteric Controls proliferation/ Activationof cAMP- Treatment of Obesity; treatment of adipokine; Fat SID;differentiation or mediated transcription in Diabetes; suppression ofbody weight proline rich acidic metabolism/ adipocytes can be gain;suppression of appetite. protein) physiology/pathology/ assayed usingmethods Hyperglycemia; Diabetes; Diabetes of adipocytes and known in theart (Berger Insipidus; Diabetes mellitus; Type 1 adipose tissue in etal., Gene 66: 1-10 diabetes; Type 2 diabetes; Insulin response todietary (1998); Cullen and resistance; Insulin deficiency; conditions.Malm, Methods in Hyperlipidemia; Hyperketonemia; Enzymol 216: 362-368Non- insulin dependent Diabetes (1992); Henthorn et al., Mellitus(NIDDM); Insulin- Proc Natl Acad Sci USA dependent Diabetes Mellitus 85:6342-6346 (1988); (IDDM); A Condition Associated Reusch et al., Mol CellWith Diabetes Including, But Not Biol 20(3): 1008-1020 Limited ToObesity, Heart Disease, (2000); and Klemm et al., Hyperglycemia,Infections, J Biol Chem 273: 917- Retinopathy, And/Or Ulcers; 923(1998)). Metabolic Disorders; Immune Disorders; Obesity; VascularDisorders; Suppression of Body Weight; Suppression of Appetite; SyndromeX. Other indications for antibodies and/or antagonists, includetreatment of weight loss; treatment of AIDS wasting; appetite stimulant;treatment of cachexia. PYY (Peptide YY), Decreases appetite; Appetiteand food intake Most Preferred: Treatment of including PYY₃₋₃₆ increasessatiety; can be can be measured Obesity; treatment of Diabetes; (aminoacid residues 31- decreases food intake. by methods known in thesuppression of body weight gain; 64 of full length PYY, art (Batterhamet al. suppression of appetite. amino acid residues 3- Nature 2002; 418:Hyperglycemia; Diabetes; Diabetes 36 of mature PYY) 650654) Insipidus;Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non- insulindependent Diabetes Mellitus (NIDDM); Insulin- dependent DiabetesMellitus (IDDM); A Condition Associated With Diabetes Including, But NotLimited To Obesity, Heart Disease, Hyperglycemia, Infections,Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;Obesity; Vascular Disorders; Suppression of Body Weight; Suppression ofAppetite; Syndrome X. Other indications for antibodies, antagonists;treatment of weight loss; treatment of AIDS wasting; appetite stimulant;treatment of cachexia. WNT10b Inhibits adipogenesis. WNT10b activity canbe Most Preferred: Treatment of measured using Obesity; suppression ofbody weight adipogenesis inhibition gain; suppression of appetite. Otherassays (Ross et al., indications; Hyperglycemia; Science 2000;289(5481): Diabetes; Diabetes Insipidus; 950-953 Diabetes mellitus; Type1 diabetes; Type 2 diabetes; Insulin resistance; Insulin deficiency;Hyperlipidemia; Hyperketonemia; Non-insulin dependent Diabetes Mellitus(NIDDM); Insulin- dependent Diabetes Mellitus (IDDM). WNT11 Promotescardiogenesis. WNT11 activity can be Treatment of Cardiovascularmeasured using assays disorders; Congestive Heart Failure; known in theart, Myocardial Infarction. including cardiogenesis assays (Eisenberg etal., Dev Dyn 1999 September; 216(1): 45-58). Herstatin Inhibits cancerHerstatin activity can be Oncology; Cancer; Solid Tumors; proliferation.measured using cell Melanoma; Malignant Melanoma; proliferation assaysRenal Cancer (e.g., Renal Cell known in the art (Doherty Carcinoma);Lung Cancer (e.g,. Non- et al., PNAS 1999; Small Cell Lung Cancer orSmall 96(19): 10869-10874. Cell Lung Cancer); Colon Cancer; BreastCancer; Liver Cancer; Prostate Cancer; Bladder Cancer; Gastric Cancer;Sarcoma; AIDS- Related Kaposi's Sarcoma; Lymphoma; T Cell Lymphoma;Cutaneous T-Cell Lymphoma; Non-Hodgkin's Lymphoma; Brain Cancer; Glioma;Glioblastoma Multiforme; Cervical Dysplasia; Leukemia; Preleukemia;Hairy Cell Leukemia; Chronic Myelogeonus Leukemia; HematologicalMalignancies; Hematological Disorders; Multiple Myeloma. Adrenomedullinstimulates vasodilation; Vasodilation can be Treatment of CongestiveHeart promotes bone growth. measured using assays Failure; Hypertension;Myocardial known in the art (Ashton Infarction; Septic Shock; et al.Pharmacology Osteoporosis; Postmenopausal 2000; 61(2): 101-105.osteoporosis; Osteopenia. The promotion of bone growth can be measuredusing assays known in the art, such as the osteoblast proliferationassay (Cornish et al. Am J Physiol 1997 December; 273(6 Pt 1): E1113-20). Nogo Receptor Receptor for the axon The promotion of axon Treatmentof Central Nervous growth inhibitor, Nogo. regeneration and growthSystem Damage; Spinal Cord Injury; can be measured using PeripheralNerve Damage; assays known in the art Neurodegenerative Diseases;(Fournier et al. Nature Parkinson's Disease; Alzheimer's 2001;409(6818): 341-346). Disease; Huntington's Disease; Amyotrophic LateralSclerosis; Progressive Supranuclear Palsy; Creutzfeld-Jacob Disease;Motor Neuron Disease. CART (Cocaine- and Inhibits food intact andAppetite and food intake Most Preferred: Treatment of Amphetamine- fatstorage; promotes can be can be measured Obesity; suppression of bodyweight Regulated Transcript) lipid oxidation. by methods known in thegain; suppression of appetite. Other art (Batterham et al. indications;Hyperglycemia; Nature 2002; 418: Diabetes; Diabetes Insipidus; 650654)Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Insulin resistance;Insulin deficiency; Hyperlipidemia; Hyperketonemia; Non-insulindependent Diabetes Mellitus (NIDDM); Insulin- dependent DiabetesMellitus (IDDM). RegIV (Colon Specific Stimulates glucose RegIV activitymay be Hyperglycemia; Diabetes; Diabetes Gene; Colon Specific uptake;increases assayed in vitro using a Insipidus; Diabetes mellitus; Type 1Protein) insulin sensitivity. [3-H]-glucose uptake diabetes; Type 2diabetes; Insulin assay. (J Biol Chem 1999 resistance; Insulindeficiency; Oct. 22; 274(43): 30864- Hyperlipidemia; Hyperketonemia;30873). Non- insulin dependent Diabetes Mellitus (NIDDM); Insulin-dependent Diabetes Mellitus (IDDM); A Condition Associated With DiabetesIncluding, But Not Limited To Obesity, Heart Disease, Hyperglycemia,Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; ImmuneDisorders; Obesity; Vascular Disorders; Suppression of Body Weight;Suppression of Appetite; Syndrome X. Cosyntropin (Cortrosyn) Syntheticcorticotropin; The activity of Endocrine; Addison's disease; (CAS-16960-16-0) stimulates the release of cosyntropin can be Cushing's syndrome;pituitary cortisol. assessed in vivo by dysfunction; acute adrenalcrisis measuring serum cortisol levels. (Frank et al. J. Am. Vet. Med.Assoc. 1998 212(10): 1569-71). Pexiganan Acetate Disrupts bacterialPexiganan acetate Treatment of Infectious Diseases; (CAS-172820-23-4)membranes. activity can be assessed Treatment of Bacterial Infections.using in vitro antibacterial assays known in the art. (Zasloff et al.,Antimicrobial Agents and Chemotherapy 1999, 43: 782-788). Pramlintide(Amylin) Slows gastric emptying; Appetite and food intake Treatment ofObesity; treatment of (CAS-151126-32-8) decreases food intake. can becan be measured Diabetes; suppression of body weight by methods known inthe gain; suppression of appetite; art (Batterham et al. treatment ofendocrine disorders; Nature 2002; 418: Hyperglycemia; Diabetes; Diabetes650654) Insipidus; Diabetes mellitus; Type 1 diabetes; Type 2 diabetes;Insulin resistance; Insulin deficiency; Hyperlipidemia; Hyperketonemia;Non- insulin dependent Diabetes Mellitus (NIDDM); Insulin- dependentDiabetes Mellitus (IDDM); A Condition Associated With DiabetesIncluding, But Not Limited To Obesity, Heart Disease, Hyperglycemia,Infections, Retinopathy, And/Or Ulcers; Metabolic Disorders; ImmuneDisorders; Obesity; Vascular Disorders; Suppression of Body Weight;Suppression of Appetite; Syndrome X. Other indications for antibodies,antagonists; treatment of weight loss; treatment of AIDS wasting;appetite stimulant; treatment of cachexia. Teriparatide (CAS- Acts inconjuction with Adenylyl cyclase Bone Disorders; Fracture prevention;52232- 67-4) calcitonin to control stimulation in rat Hypercalcemia;Malignant calcium and phosphate osteosarcoma cells, hypercalcemia;Osteoporosis; Paget's metabolism; elevates ovariectomized rat modeldisease; Osteopenia, blood calcium level; of osteoporosis: IUBMBOsteoclastogenesis; osteolysis; stimulates the activity Life 2000February; 49(2): osteomyelitis; osteonecrosis; of osteocytes; enhances131-5 periodontal bone loss; osteoarthritis; absorption of Ca+/Pirheumatoid arthritis; osteopetrosis; from small intestine periodontal,lytic, or metastatic bone into blood; promotes disease; osteoclastdifferentiation reabsorption of Ca+ and inhibition; bone disorders; boneinhibits Pi by kidney healing and regeneration. tubules. Terlipressin(triglycyl Analog of vasopressin; Terlipressin activity can Varicealhemorrhage; cirrhosis; lycine vasopressin) induces be measured usingassays portal hypertension; hepatorenal (CAS-14636- 12-5)vasoconstriction. of vasoconstriction, such syndrome; Blood-relateddisorders as the isolated arterial ring preparation. (Landstrom et al.,Hum Reprod 1999 January; 14(1): 151-5), Ularitide (CAS-118812-Stimulates natriuresis, Ularitide activity can be Excretory disorders;Acute renal 69-4) diuresis, and assessed by measuring failure; asthma;congestive heart vasodilation. cGMP accumulation in failure;hypertension; pulmonary rat renal cells. (Valentin hypertension;cardiovascular et al., Hypertension 1993 disorders April; 21(4): 432-8).Aprotinin (Trasylol) Serine protease Inhibition of thrombin- Inhibitionof fibrinolysis; reduction (CAS-9087-70-1; CAS- inhibitor; attenuatesinduced platelet of blood loss during surgery; 11061-94-2; CAS- SystemicInflammatory aggregation can be Treatment of Inflammation and 12407-79-3) Response, fibrinolysis measured using methods immune Disorders.and thrombin-induced known in the art. (Poullis platelet aggregation. etal., J Thorac Cardiovasc Surg 2000 August; 120(2): 370-8). Aspartocin(CAS-4117- Antibacteria Aspartocin activity can Treatment of InfectiousDiseases; 65-1; CAS- 1402-89-7) be assessed using in vitro treatment ofbacterial infections. antibacterial assays known in the art. (Zasloff etal., Antimicrobial Agents and Chemotherapy 1999, 43: 782-788).Calcitonin (Calcimar) Regulates levels of Hypocalcemic RatMusculoskeletal; Osteroporosis; (CAS-21215-62-3) calcium and phosphateBioassay, bone resorbing Paget's disease; hypercalcemia; Bone in serum;causes a assay and the pit assay, Disorders; Fracture prevention;reduction in serum CT receptor binding Malignant hypercalcemia;calcium--an effect assay, CAMP stimulation Osteopenia,Osteoclastogenesis; opposite to that of assay: J Bone Miner Resosteolysis; osteomyelitis; human parathyroid 1999 August; 14(8):osteonecrosis; periodontal bone loss; hormone. 1425-31 osteoarthritis;rheumatoid arthritis; osteopetrosis; periodontal, lytic, or metastaticbone disease; osteoclast differentiation inhibition; bone disorders;bone healing and regeneration. Carperitide (HANP; Stimulatesnatriuresis, Carperitide activity can Treatment of Heart Failure;recombinant human diuresis, and be assessed in vitro by Cardiovasculardisorders; Respiratory atrial natriuretic peptide) vasodilation.measuring cGMP disorders; Acute respiratory distress (CAS-89213- 87-6)accumulation in a number syndrome. of cell lines, including PC12 cellsand cultured human glomerular cells. (Medvede et al., Life Sci 2001 Aug.31; 69(15): 1783-90; Green et al., J Am Soc Nephrol 1994 October; 5(4):1091-8). Desirudin (recombinant Inhibits thrombin; Desirudin activitycan be Blood-related disorder; Thrombosis; hirudin; Revasc) (CAS-inhibits blood clotting. assessed using blood thrombocytopenia;hemorrhages. 120993- 53-5) clotting assays known in the art, such as invitro platelet aggregation assays. (Glusa, Haemostasis 1991; 21 Suppl 1:116-20). Emoctakin (interleukin proinflammatory Treatment ofInflammation, Immune 8) (CAS-142298- 00-8) cytokine disorders. RSVinfection. Felypressin (CAS-56- Derivative of Felypressin Treatment ofpain; to induce local 59-7) Vasopressin; Stimulates vasoconstrictionactivity anesthesia. vasoconstriction; can be measured using Induceslocal assays of anesthesia. vasoconstriction, such as the isolatedarterial ring preparation. (Landstrom et al., Hum Reprod 1999 January;14(1): 151-5). Glucagon (CAS-16941- Induces hyperglycemia. Glucagonactivity may be Hypoglycemia; Diabetes; Diabetes 32-5) assayed in vitrousing a Insipidus; Diabetes mellitus; Type 1 [3-H]-glucose uptakediabetes; Type 2 diabetes; Insulin assay. (J Biol Chem 1999 resistance;Insulin deficiency; Oct. 22; 274(43): 30864- Hyperlipidemia;Hyperketonemia; 30873). Non- insulin dependent Diabetes Mellitus(NIDDM); Insulin- dependent Diabetes Mellitus (IDDM); A ConditionAssociated With Diabetes Including, But Not Limited To Obesity, HeartDisease, Hyperglycemia, Infections, Retinopathy, And/Or Ulcers;Metabolic Disorders; Immune Disorders; Obesity; Vascular Disorders;Suppression of Body Weight; Suppression of Appetite; Syndrome X;Endocrine disorders. Nagrestipen (CAS- Inflammation; Immune 166089-33-4) Pentigetide (Pentyde) Respiratory; Allergy; Immune (CAS-62087-72-3) Proinsulin (CAS-67422- Stimulates glucose Insulin activity may beHyperglycemia; Diabetes; Diabetes 14-4) uptake and promotes assayed invitro using a Insipidus; Diabetes mellitus; Type 1 glycogenesis and[3-H]-glucose uptake diabetes; Type 2 diabetes; Insulin lipogenesis.assay. (J Biol Chem 1999 resistance; Insulin deficiency; Oct. 22;274(43): 30864- Hyperlipidemia; Hyperketonemia; 30873). Non- insulindependent Diabetes Mellitus (NIDDM); Insulin- dependent DiabetesMellitus (IDDM); A Condition Associated With Diabetes Including, But NotLimited To Obesity, Heart Disease, Hyperglycemia, Infections,Retinopathy, And/Or Ulcers; Metabolic Disorders; Immune Disorders;Obesity; Vascular Disorders; Suppression of Body Weight; Suppression ofAppetite; Syndrome X. Becaplermin (Regranex; Promotes wound Becaplerminactivity can Stimulate Epithelial Cell recombinant PDGF-BB) healing. beassessed using animal Proliferation; Stimulate Basal (CAS-165101- 51-9)wound healing models Keratinocytes; Promote Wound known in the art.(Saba et Healing; Stimulate Hair Follicle al., Ann Plast Surg 2002Production; Healing Of Dermal July; 49(1): 62-6). Wounds. Wound Healing;Eye Tissue Wounds, Dental Tissue Wounds, Oral Cavity Wounds, DiabeticUlcers, Dermal Ulcers, Cubitus Ulcers, Arterial Ulcers, Venous StasisUlcers, Burns Resulting From Heat Exposure Or Chemicals, or OtherAbnormal Wound Healing Conditions such as Uremia, Malnutrition, VitaminDeficiencies or Complications Associated With Systemic Treatment WithSteroids, Radiation Therapy or Antineoplastic Drugs or Antimetabolites;Promote Dermal Reestablishment Subsequent To Dermal Loss; Increase theAdherence Of Skin Grafts To A Wound Bed; Stimulate Re- Epithelializationfrom The Wound Bed; To Promote Skin Strength; Improve The Appearance OfAged Skin; Proliferate Hepatocytes, Lung, Breast, Pancreas, Stomach,Bladder, Small Intestine, Large Intestine; Sebocytes, Hair Follicles,Type II Pneumocytes, Mucin-Producing Goblet Cells, or Other EpithelialCells, Endothelial Cells, Keratinocytes, or Basal Keratinocytes (andTheir Progenitors) Contained Within The Skin, Lung, Liver, Bladder, Eye,Salivary Glands, or Gastrointestinal Tract; Reduce The Side Effects OfGut Toxicity That Result From Radiation, Chemotherapy Treatments OrViral Infections; Cytoprotector, especially of the Small IntestineMucosa or Bladder; Mucositis (Mouth Ulcers); Regeneration Of Skin; Fulland/or Partial Thickness Skin Defects, including Burns, (e.g.,Repopulation Of Hair Follicles, Sweat Glands, And Sebaceous Glands);Psoriasis; Epidermolysis Bullosa; Blisters; Gastric and/or DoudenalUlcers; Reduce Scarring; Inflamamatory Bowel Diseases; Crohn's Disease;Ulcerative Colitis; Gut Toxicity; Lung Damage; Repair Of Alveoli And/orBrochiolar Epithelium; Acute Or Chronic Lung Damage; Emphysema, ARDS;Inhalation Injuries; Hyaline Membrane Diseases; Infant RespiratoryDistress Syndrome; Bronchopulmonary Displasia In Premature Infants;Fulminant Liver Failure; Cirrhosis, Liver Damage caused by ViralHepatitis and/or Toxic Substances; Diabetes Mellitus; Inflammation;Cancer; Digestive disorders. Ghrelin Stimulates release of Appetite andfood Endocrine; loss of body weight; loss of (Genbank growth hormoneintake can be can be body weight associated with cancer or Accession No.from anterior measured by methods anorexia nervosa; loss of appetite;AB029434) pituitary. Stimulates known in the art excessive appetite;body weight gain; appetite and reduces (Batterham et al. Obesity;Diabetes; Acromegaly; fat burning. Nature 2002; Growth 418: 650654)failure; Growth hormone deficiency; Growth failure and growthretardation Prader-Willi syndrome in children 2 years or older; Growthdeficiencies; Growth failure associated with chronic renalinsufficiency; Postmenopausal osteoporosis; burns; cachexia; cancercachexia; dwarfism; metabolic disorders; obesity; renal failure;Turner's Syndrome, pediatric and adult; fibromyalgia; fracturetreatment; frailty, AIDS wasting Ghrelin - binding Inhibits growthAppetite and food intake Endocrine; Obesity; Diabetes; body antibodyincluding hormone release in can be can be measured weight gain;excessive appetite; loss antibody fragment, or response to Ghrelin; bymethods known in the of appetite; loss of body weight. dominant-negative form inhibits increase in art (Batterham et al. of Ghrelinappetite. Nature 2002; 418: 650654) receptor NOGO-66 Neurodegenerativedisorders; spinal peptide fragment cord injury; neuronal injury; brain(Genbank Accession trauma; stroke; multiple sclerosis; No. NP_008939(amino demyelinating disorders; neural acids 62-101)) activity andneurological diseases; neural cell (e.g., neuron, glial cell, andschwann cell) regeneration and/or growth Gastric inhibitory Increasesnutrient Nutrient uptake and Most Preferred: loss of body weight,polypeptide (GIP), uptake and tryglyceride tryglyceride AIDS wasting,cachexia, and loss of including GIP fragments accumulation inaccumulation can be appetite. Other: Obesity; Diabetes; (GenbankAccession adipocytes, which leads measured by methods insulinresistance; body weight gain; No. NM_004123) to obesity and insulindescribed in Miyawaki et excessive appetite. resistance. al., Nat.Medicine, 2002, Vol 8(7): 738-742, Gastric inhibitory Increased use offat as Fat utilization as an Obesity; Diabetes; Insulin resistance;polypeptide antibody, or predominant energy energy source can be bodyweight gain. antibody fragments source; decreased measured as describedin accumulation of fat in Miyawaki et al., Nat. adipocytes. Medicine,2002, Vol 8(7): 738-742, Gastric inhibitory Increased use of fat as Fatutilization as an Most preferred: Obesity; Diabetes; peptide receptor orpredominant energy energy source can be body weight gain; excessiveappetite; receptor fragments or source; decreased measured as describedin insulin resistance. Other: loss of body variants includingaccumulation of fat in Miyawaki et al., Nat. weight, AIDS wasting, lossof soluble fragments or adipocytes. Medicine, 2002, Vol appetite.variants (Genbank 8(7): 738-742. Accession Number NM_000164) POMCActivity of POMC- Preferred: resistance to stress; anti-(proopiomelanocortin), derived fragments are inflammatory activity;analgesic including fragments or diverse, and well- activity; increasedskin pigmentation; variants (such as, for known in the art. See,increased protein catabolism; example, alpha- for example, Hadley etincreased gluconeogenesis; obesity; melanocyte stimulating al., Ann NYAcad Sci diabetes. Other: decreased protein hormone, αMSH, 1999 Oct. 20;885: 1-21; catabolism, decreased skin gamma melanocyte Dores, Prog ClinBiol pigmentation, Addison's disease, stimulating hormone, Res 1990;342: 22-7; Cushing's syndrome γMSH, beta- melanocyte Blalock, Ann NYAcad stimulating hormone, Sci. 1999 Oct. 20; 885: βMSH, 161-72).adrenocorticotropin, ACTH, beta- endorphin, met- enkephalin) (GenbankAccession No. NM_000930) HP 467, HP228 (U.S. See U.S. Pat. No. See U.S.Pat. No. Resistance to stress; anti- Pat. No. 6,350,430) 6,350,4306,350,430 inflammatory activity; analgesic activity; increased skinpigmentation; increased protein catabolism; increased gluconeogenesis.NDP (U.S. Pat. No. See U.S. Pat. No. See U.S. Pat. No. Resistance tostress; anti- 6,350,430) 6,350,430 6,350,430 inflammatory activity;analgesic activity; increased skin pigmentation; increased proteincatabolism; increased gluconeogenesis. Interleukin-21 (IL-21)Immunomodulator; IL-21 activity can be Autoimmune disorders; inhibitsinterferon assessed by measuring Inflammatory disorders; Treatment ofgamma production by interferon gamma Psoriasis; Rheumatoid Arthritis;Th1 cells, production in Th1 cells. Inflammatory bowel disease. (Wursteret al.,: J Exp Med 2002 Oct. 7; 196(7): 969-77) Interleukin-4 (IL-4)Immunomodulator; IL-4 activity can be Treatment of Psoriasis; Autoimmunepromotes the assessed by measuring disorders; Rheumatoid Arthritis;differentiation of T cells Th1/Th2 cytokine Inflammatory bowel disease;into Th2 phenotype. responses of isolated Inflammatory disorders. spleencells in vitro. (Waltz et al., Horm Metab Res 2002 October; 34(10):561-9). Osteoclast Inhibitory Inhibits osteoclast Osteoclast InhibitoryTreatment of Bone Disorders; Lectin (OCIL) formation. Lectin activitycan be Osteoporosis; Fracture prevention; assessed using osteoclastHypercalcemia; Malignant formation assays known hypercalcemia; Paget'sdisease; in the art. (Zhou et al., J Osteopenia, Osteoclastogenesis;Biol Chem 2002 Dec. 13; osteolysis; osteomyelitis; 277(50): 48808-15)osteonecrosis; periodontal bone loss; osteoarthritis; rheumatoidarthritis; osteopetrosis; periodontal, lytic, or metastatic bonedisease; osteoclast differentiation inhibition; bone healing andregeneration. PCSK9 Inhibitor Inhibits the interaction Further LDLlowering Treatment of coronary heart disease. of PCSK9 with LDL throughtargeting PCSK9 Receptor. for coronary artery disease. (Cao et al.Endocrine, Metabolic & Immune Disorders-Drug Targets 2008, 8, 238-243)

Functional Activity:

“A polypeptide having functional activity” refers to a polypeptidecapable of displaying one or more known functional activities associatedwith the full-length, pro-protein, and/or mature form of a cargopolypeptide. Such functional activities include, but are not limited to,biological activity, antigenicity [ability to bind (or compete with apolypeptide for binding) to an anti-polypeptide antibody],immunogenicity (ability to generate antibody which binds to a specificpolypeptide described herein), ability to form multimers withpolypeptides described herein, and ability to bind to a receptor orligand for a polypeptide. In certain embodiments, the functionalactivity includes the ability to improve the expression and stability ofa partner protein.

“A polypeptide having biological activity” refers to a polypeptideexhibiting activity similar to, but not necessarily identical to, anactivity of a therapeutic protein described herein, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide described herein (i.e., the candidate polypeptide willexhibit greater activity or not more than about 25-fold less, or notmore than about tenfold less activity, or not more than about three-foldless activity relative to a polypeptide described herein, or presentedin Table 2).

In certain embodiments, a heteromultimer described herein has at leastone biological and/or therapeutic activity associated with the cargomolecule when said cargo molecule is not linked to the transporterpolypeptide. In certain embodiments, a heteromultimer described hereinhas at least one biological and/or therapeutic activity associated withthe cargo polypeptide when said cargo polypeptide is not linked to thetransporter polypeptide. In certain embodiments, a heteromultimericprotein described herein has at least one biological and/or therapeuticactivity associated with the cargo polypeptide portion (or fragment orvariant thereof) when said cargo polypeptide is not linked to thealbumin or alloalbumin based polypeptide.

The heteromultimeric proteins described herein can be assayed forfunctional activity (e.g., biological activity) using or routinelymodifying assays known in the art, as well as assays described herein.Additionally, one of skill in the art may routinely assay fragments of aprotein corresponding to a cargo protein portion of an albumin oralloalbumin based monomeric polypeptide, for activity using assaysreferenced in its corresponding row of Table 2 (e.g., in column 3 ofTable 2). In certain embodiments, are assay of fragments of an albuminprotein corresponding to an albumin protein portion of a heteromultimer,for activity using assays known in the art and/or as described in theExamples section below.

For example, in one embodiment where one is assaying for the ability ofa heteromultimeric protein described herein to bind or compete with aCargo polypeptide for binding to an anti-Cargo polypeptide antibodyand/or anti-albumin antibody, various immunoassays known in the art canbe used, including but not limited to, competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitation reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labeled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention.

In certain embodiments, where a binding partner (e.g., a receptor or aligand) is identified for a cargo molecule comprised by a heteromultimerdescribed herein, binding to that binding partner by a heteromultimerdescribed herein is assayed, e.g., by means well-known in the art, suchas, for example, reducing and non-reducing gel chromatography, proteinaffinity chromatography, and affinity blotting. See generally, Phizickyet al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, theability of physiological correlates of a heteromultimeric protein tobind to a substrate(s) of polypeptides corresponding to the cargoprotein portion of the heteromultimer can be routinely assayed usingtechniques known in the art.

Biological Activities

In certain embodiments, heteromultimers described herein, are used inassays to test for one or more biological activities. If aheteromultimer exhibits an activity in a particular assay, it is likelythat at least one cargo protein comprised by one or more monomers of theheteromultimer is implicated in the diseases associated with thebiological activity. Thus, the heteromultimer is of use in a treatmentof the associated disease.

In certain embodiments, provided is a method of treating a disease ordisorder comprising administering to a patient in which such treatment,prevention or amelioration is desired, a heteromultimer describedherein, in an amount effective to treat, prevent or ameliorate thedisease or disorder.

Provided herein are monomeric albumin or alloalbumin based fusionproteins produced by a cell, wherein said proteins are encoded bypolynucleotides, wherein said monomeric proteins comprise at least onecargo protein, and an albumin or alloalbumin derived polypeptide, suchthat said monomers form heteromultimers in solution. In certainembodiments, when the polynucleotides are used to express the encodedprotein from a cell, the cell's natural secretion and processing stepsproduces a protein that lacks at least one signal sequence. The specificamino acid sequence of the signal sequence is well known in the art.

In certain embodiments, heteromultimers described herein are used in thediagnosis, prognosis, prevention and/or treatment of diseases and/ordisorders of the endocrine system. In some embodiments, heteromultimersdescribed herein are used in the diagnosis, prognosis, prevention and/ortreatment of diseases and/or disorders of the nervous system.

In certain embodiments, heteromultimers described herein are used in thediagnosis, prognosis, prevention and/or treatment of diseases and/ordisorders of the immune system. In certain embodiments, heteromultimersdescribed herein are used in the diagnosis, prognosis, prevention and/ortreatment of diseases and/or disorders of the respiratory system.

In certain embodiments, heteromultimers described herein are used in thediagnosis, prognosis, prevention and/or treatment of diseases and/ordisorders of the cardiovascular system. In some embodiments,heteromultimers described herein are used in the diagnosis, prognosis,prevention and/or treatment of diseases and/or disorders of thereproductive system.

In certain embodiments, heteromultimers described herein are used in thediagnosis, prognosis, prevention and/or treatment of diseases and/ordisorders of the digestive system. In certain embodiments,heteromultimer proteins described herein are used in the diagnosis,prognosis, prevention and/or treatment of diseases or disorders relatingto the blood.

In certain embodiments, heteromultimers described herein are used in thediagnosis and/or prognosis of diseases and/or disorders associated withat least one tissue(s) in which at least one gene of interest isexpressed, wherein a heteromultimer described herein comprises a cargomolecule that binds said at least one gene of interest.

In some embodiments, heteromultimers described herein and/orpolynucleotides encoding the albumin/alloalbumin based monomers thatassociate to form heteromultimers described herein, are used in thediagnosis, detection and/or treatment of diseases and/or disordersassociated with activities that include, but are not limited to,prohormone activation, neurotransmitter activity, cellular signaling,cellular proliferation, cellular differentiation, and cell migration.

Therapeutic Uses:

In an aspect, heteromultimers described herein are directed toantibody-based therapies which involve administering heteromultimersdescribed comprising cargo polypeptide(s) which is an antibody, afragment or variant of an antibody, to a patient for treating one ormore of the disclosed diseases, disorders, or conditions. Therapeuticcompounds described herein include, but are not limited to,heteromultimers described herein, nucleic acids encoding heteromultimersdescribed herein.

In a specific embodiment, are antibody-based therapies which involveadministering heteromultimers described herein comprising at least afragment or variant of an antibody to a patient for treating one or morediseases, disorders, or conditions, including but not limited to: neuraldisorders, immune system disorders, muscular disorders, reproductivedisorders, gastrointestinal disorders, pulmonary disorders,cardiovascular disorders, renal disorders, proliferative disorders,and/or cancerous diseases and conditions, and/or as described elsewhereherein.

A summary of the ways in which the heteromultimer proteins of theinvention comprising at least a fragment or variant of an antibody areused therapeutically includes binding locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theheteromultimers described herein for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The heteromultimers described herein, comprising at least a fragment orvariant of an antibody may be administered alone or in combination withother types of treatments (e.g., radiation therapy, chemotherapy,hormonal therapy, immunotherapy and anti-tumor agents). Generally,administration of products of a species origin or species reactivity (inthe case of antibodies) that is the same species as that of the patientis preferred. Thus, in an embodiment, human antibodies, fragmentsderivatives, analogs, or nucleic acids, are administered to a humanpatient for therapy or prophylaxis.

Gene Therapy:

In a specific embodiment, nucleic acids comprising sequences encodingheteromultimer proteins described herein are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a protein, by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this embodiment of theinvention, the nucleic acids produce their encoded protein that mediatesa therapeutic effect. Any of the methods for gene therapy available inthe art can be used.

Demonstration of Therapeutic or Prophylactic Activity:

The heteromultimers or pharmaceutical compositions described herein aretested in vitro, and then in vivo for the desired therapeutic orprophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered aheteromultimer, and the effect of such heteromultimer upon the tissuesample is observed.

Therapeutic/Prophylactic Administration and Composition

Provided are methods of treatment, inhibition and prophylaxis byadministration to a subject of an effective amount of a heteromultimeror pharmaceutical composition described herein. In an embodiment, theheteromultimer is substantially purified (e.g., substantially free fromsubstances that limit its effect or produce undesired side-effects). Incertain embodiments, the subject is an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andin certain embodiments, a mammal, and most preferably human.

Various delivery systems are known and can be used to administer aheteromultimer formulation described herein, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the compound, receptor-mediated endocytosis (see, e.g., Wuand Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleicacid as part of a retroviral or other vector, etc. Methods ofintroduction include but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, in certain embodiments, it is desirableto introduce the heteromultimer compositions described herein into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it is desirable to administer theheteromultimers, or compositions described herein locally to the area inneed of treatment; this may be achieved by, for example, and not by wayof limitation, local infusion during surgery, topical application, e.g.,in conjunction with a wound dressing after surgery, by injection, bymeans of a catheter, by means of a suppository, or by means of animplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as silastic membranes, or fibers.Preferably, when administering a protein, including an antibody, of theinvention, care must be taken to use materials to which the protein doesnot absorb.

In another embodiment, the heteromultimers or composition can bedelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

In yet another embodiment, the heteromultimers or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,e.g., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment comprising a nucleic acid encoding aheteromultimer described herein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

Also provided herein are pharmaceutical compositions. Such compositionscomprise a therapeutically effective amount of a compound, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In certain embodiments, the composition comprising the heteromultimer isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to human beings.Typically, compositions for intravenous administration are solutions insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic such aslignocaine to ease pain at the site of the injection. Generally, theingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientsmay be mixed prior to administration.

In certain embodiments, the compositions described herein are formulatedas neutral or salt forms. Pharmaceutically acceptable salts includethose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxide isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the composition described herein which will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a Therapeuticprotein can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses areextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Methods of Recombinant and Synthetic Production of HeteromultimerProteins:

In certain embodiments are heteromultimers produced as recombinantmolecules by secretion from yeast, a microorganism such as a bacterium,or a human or animal cell line. In embodiments, the polypeptides aresecreted from the host cells.

Embodiments include a cell, such as a yeast cell transformed to expressa heteromultimer protein described herein. In addition to thetransformed host cells themselves, are provided culture of those cells,preferably a monoclonal (clonally homogeneous) culture, or a culturederived from a monoclonal culture, in a nutrient medium. If thepolypeptide is secreted, the medium will contain the polypeptide, withthe cells, or without the cells if they have been filtered orcentrifuged away. Many expression systems are known and may be used,including bacteria (for example E. coli and Bacillus subtilis), yeasts(for example Saccharomyces cerevisiae, Kluyveromyces lactis and Pichiapastoris, filamentous fungi (for example Aspergillus), plant cells,animal cells and insect cells.

A heteromultimer described herein is produced in conventional ways, forexample from a coding sequence inserted in the host chromosome or on afree plasmid. The yeasts are transformed with a coding sequence for thedesired protein in any of the usual ways, for example electroporation.Methods for transformation of yeast by electroporation are disclosed inBecker & Guarente (1990) Methods Enzymol. 194, 182.

Successfully transformed cells, i.e., cells that contain a DNA constructof the present invention, can be identified by well known techniques.For example, cells resulting from the introduction of an expressionconstruct can be grown to produce the desired polypeptide. Cells can beharvested and lysed and their DNA content examined for the presence ofthe DNA using a method such as that described by Southern (1975) J. Mol.Biol. 98, 503 or Berent et al. (1985) Biotech. 3, 208. Alternatively,the presence of the protein in the supernatant can be detected usingantibodies.

Useful yeast plasmid vectors include pRS403-406 and pRS413-416 and aregenerally available from Stratagene Cloning Systems, La Jolla, Calif.92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are YeastIntegrating plasmids (YIps) and incorporate the yeast selectable markersHIS3, 7RP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromereplasmids (Ycps).

A variety of methods have been developed to operably link DNA to vectorsvia complementary cohesive termini. For instance, complementaryhomopolymer tracts can be added to the DNA segment to be inserted to thevector DNA. The vector and DNA segment are then joined by hydrogenbonding between the complementary homopolymeric tails to formrecombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. The DNAsegment, generated by endonuclease restriction digestion, is treatedwith bacteriophage T4 DNA polymerase or E. coli DNA polymerase 1,enzymes that remove protruding, . . . -single-stranded termini withtheir 3′ 5′-exonucleolytic activities, and fill in recessed 3′-ends withtheir polymerizing activities.

The combination of these activities therefore generates blunt-ended DNAsegments. The blunt-ended segments are then incubated with a large molarexcess of linker molecules in the presence of an enzyme that is able tocatalyze the ligation of blunt-ended DNA molecules, such asbacteriophage T4 DNA ligase. Thus, the products of the reaction are DNAsegments carrying polymeric linker sequences at their ends. These DNAsegments are then cleaved with the appropriate restriction enzyme andligated to an expression vector that has been cleaved with an enzymethat produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sitesare commercially available from a number of sources includingInternational Biotechnologies Inc, New Haven, Conn., USA.

Exemplary genera of yeast contemplated to be useful in the practice ofthe present invention as hosts for expressing the albumin, fusionproteins are Pichua (formerly classified as Hansenula), Saccharomyces,Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora,Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces,Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora,Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus,Sporidiobolus, Endomycopsis, and the like. Preferred genera are thoseselected from the group consisting of Saccharomyces,Schizosaccharomyces, Kluyveromyces, Pichia and Torulaspora. Examples ofSaccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.

Examples of Kluyveromyces spp. are K. fragilis, K. lactis and K.marxianus. A suitable Torulaspora species is T. delbrueckii. Examples ofPichia (Hansenula) spp. are P. angusta (formerly H. polymorpha), P.anomala (formerly H. anomala) and P. pastoris. Methods for thetransformation of S. cerevisiae are taught generally in EP 251 744, EP258 067 and WO 90/01063, all of which are incorporated herein byreference.

Preferred exemplary species of Saccharomyces include S. cerevisiae, S.italicus, S. diastaticus, and Zygosaccharomyces rouxii. Preferredexemplary species of Kluyveromyces include K. fragilis and K. lactis.Preferred exemplary species of Hansenula include H. polymorpha (nowPichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata.Additional preferred exemplary species of Pichia include P. pastoris.Preferred exemplary species of Aspergillus include A. niger and A.nidulans. Preferred exemplary species of Yarrowia include Y. lipolytica.Many preferred yeast species are available from the ATCC. For example,the following preferred yeast species are available from the ATCC andare useful in the expression of albumin fusion proteins: Saccharomycescerevisiae, Hansen, teleomorph strain BY4743 yap3 mutant (ATCC AccessionNo. 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743hsp150 mutant (ATCC Accession No. 4021266); Saccharomyces cerevisiaeHansen, teleomorph strain BY4743 pmt1 mutant (ATCC Accession No.4023792); Saccharomyces cerevisiae Hansen, teleomorph (ATCC AccessionNos. 20626; 44773; 44774; and 62995); Saccharomyces diastaticus Andrewset Gilliland ex van der Walt, teleomorph (ATCC Accession No. 62987);Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph (ATCCAccession No. 76492); Pichia angusta (Teunisson et al.) Kurtzman,teleomorph deposited as Hansenula polymorpha de Morais et Maia,teleomorph (ATCC Accession No. 26012); Aspergillus niger van Tieghem,anamorph (ATCC Accession No. 9029); Aspergillus niger van Tieghem,anamorph (ATCC Accession No. 16404); Aspergillus nidulans (Eidam)Winter, anamorph (ATCC Accession No. 48756); and Yarrowia lipolytica(Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC AccessionNo. 201847).

Suitable promoters for S. cerevisiae include those associated with thePGKI gene, GAL1 or GAL10 genes, CYCI, PH05, TRP1, ADH1, ADH2, the genesfor glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, triose phosphate isomerase,phosphoglucose isomerase, glucokinase, alpha-mating factor pheromone, [amating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDIpromoter, and hybrid promoters involving hybrids of parts of 5′regulatory regions with parts of 5′ regulatory regions of otherpromoters or with upstream activation sites (e.g. the promoter ofEP-A-258 067).

Convenient regulatable promoters for use in Schizosaccharomyces pombeare the thiamine-repressible promoter from the nmt gene as described byMaundrell (1990) J. Biol. Chem. 265, 10857-10864 and the glucoserepressible jbpl gene promoter as described by Hoffman & Winston (1990)Genetics 124, 807-816.

Methods of transforming Pichia for expression of foreign genes aretaught in, for example, Cregg et al. (1993), and various Phillipspatents (e.g. U.S. Pat. No. 4,857,467, incorporated herein byreference), and Pichia expression kits are commercially available fromInvitrogen BV, Leek, Netherlands, and Invitrogen Corp., San Diego,Calif. Suitable promoters include AOX1 and AOX2. Gleeson et al. (1986)J. Gen. Microbiol. 132, 3459-3465 include information on Hansenulavectors and transformation, suitable promoters being MOX1 and FMD1;whilst EP 361 991, Fleer et al. (1991) and other publications fromRhone-Poulenc Rorer teach how to express foreign proteins inKluyveromyces spp., a suitable promoter being PGKI.

The transcription termination signal is preferably the 3′ flankingsequence of a eukaryotic gene which contains proper signals fortranscription termination and polyadenylation. Suitable 3′ flankingsequences may, for example, be those of the gene naturally linked to theexpression control sequence used, i.e. may correspond to the promoter.Alternatively, they may be different in which case the terminationsignal of the S. cerevisiae ADHI gene is preferred.

In certain embodiments, the desired heteromultimer protein is initiallyexpressed with a secretion leader sequence, which may be any leadereffective in the yeast chosen. Leaders useful in S. cerevisiae includethat from the mating factor alpha polypeptide (MFα-1) and the hybridleaders of EP-A-387 319. Such leaders (or signals) are cleaved by theyeast before the mature albumin is released into the surrounding medium.Further such leaders include those of S. cerevisiae invertase (SUC2)disclosed in JP 62-096086 (granted as 911036516), acid phosphatase(PH05), the pre-sequence of MFα-1, 0 glucanase (BGL2) and killer toxin;S. diastaticus glucoarnylase Il; S. carlsbergensis α-galactosidase(MEL1); K. lactis killer toxin; and Candida glucoarnylase.

Provided are vectors containing a polynucleotide encoding aheteromultimer protein described herein, host cells, and the productionof the heteromultimer proteins by synthetic and recombinant techniques.The vector may be, for example, a phage, plasmid, viral, or retroviralvector. Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

In certain embodiments, the polynucleotides encoding heteromultimerproteins described herein are joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

In certain embodiments, the polynucleotide insert is operatively linkedto an appropriate promoter, such as the phage lambda PL promoter, the E.coli lac, trp, phoA and rac promoters, the SV40 early and late promotersand promoters of retroviral LTRs, to name a few. Other suitablepromoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination, and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the transcripts expressed by theconstructs will preferably include a translation initiating codon at thebeginning and a termination codon (UAA, UGA or UAG) appropriatelypositioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418, glutamine synthase, or neomycin resistance for eukaryotic cellculture, and tetracycline, kanamycin or ampicillin resistance genes forculturing in E. coli and other bacteria. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris(ATCC Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A; pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

In one embodiment, polynucleotides encoding a heteromultimer proteindescribed herein are fused to signal sequences that will direct thelocalization of a protein of the invention to particular compartments ofa prokaryotic or eukaryotic cell and/or direct the secretion of aprotein of the invention from a prokaryotic or eukaryotic cell. Forexample, in E. coli, one may wish to direct the expression of theprotein to the periplasmic space. Examples of signal sequences orproteins (or fragments thereof) to which the heteromultimeric proteinsare fused in order to direct the expression of the polypeptide to theperiplasmic space of bacteria include, but are not limited to, the pelBsignal sequence, the maltose binding protein (MBP) signal sequence, MBP,the ompA signal sequence, the signal sequence of the periplasmic E. coliheat-labile enterotoxin B-subunit, and the signal sequence of alkalinephosphatase. Several vectors are commercially available for theconstruction of fusion proteins which will direct the localization of aprotein, such as the pMAL series of vectors (particularly the pMAL-.rho.series) available from New England Biolabs. In a specific embodiment,polynucleotides albumin fusion proteins of the invention may be fused tothe pelB pectate lyase signal sequence to increase the efficiency ofexpression and purification of such polypeptides in Gram-negativebacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents ofwhich are herein incorporated by reference in their entireties.

Examples of signal peptides that are fused to a heteromultimeric proteinin order to direct its secretion in mammalian cells include, but are notlimited to, the MPIF-1 signal sequence (e.g., amino acids 1-21 ofGenBank Accession number AAB51134), the stanniocalcin signal sequence(MLQNSAVLLLLVISASA), and a consensus signal sequence(MPTWAWWLFLVLLLALWAPARG). A suitable signal sequence that may be used inconjunction with baculoviral expression systems is the gp67 signalsequence (e.g., amino acids 1-19 of GenBank Accession Number AAA72759).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availability of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene. A glutamine synthase expression system and componentsthereof are detailed in PCT publications: WO87/04462; WO86/05807;WO89/10036; WO89/10404; and WO91/06657, which are hereby incorporated intheir entireties by reference herein. Additionally, glutamine synthaseexpression vectors can be obtained from Lonza Biologics, Inc.(Portsmouth, N.H.). Expression and production of monoclonal antibodiesusing a GS expression system in murine myeloma cells is described inBebbington et al., Bio/technology 10:169(1992) and in Biblia andRobinson Biotechnol. Prog. 11:1(1995) which are herein incorporated byreference.

Also provided are host cells containing vector constructs describedherein, and additionally host cells containing nucleotide sequences thatare operably associated with one or more heterologous control regions(e.g., promoter and/or enhancer) using techniques known of in the art.The host cell can be a higher eukaryotic cell, such as a mammalian cell(e.g., a human derived cell), or a lower eukaryotic cell, such as ayeast cell, or the host cell can be a prokaryotic cell, such as abacterial cell. A host strain may be chosen which modulates theexpression of the inserted gene sequences, or modifies and processes thegene product in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thusexpression of the genetically engineered polypeptide may be controlled.Furthermore, different host cells have characteristics and specificmechanisms for the translational and post-translational processing andmodification (e.g., phosphorylation, cleavage) of proteins. Appropriatecell lines can be chosen to ensure the desired modifications andprocessing of the foreign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of theinvention into the host cell can be effected by calcium phosphatetransfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,or other methods. Such methods are described in many standard laboratorymanuals, such as Davis et al., Basic Methods In Molecular Biology(1986). It is specifically contemplated that the polypeptides of thepresent invention may in fact be expressed by a host cell lacking arecombinant vector.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., the coding sequence corresponding to a Cargopolypeptide is replaced with a heteromultimer protein corresponding tothe Cargo polypeptide), and/or to include genetic material. The geneticmaterial operably associated with the endogenous polynucleotide mayactivate, alter, and/or amplify endogenous polynucleotides.

In addition, techniques known in the art may be used to operablyassociate heterologous polynucleotides (e.g., polynucleotides encodingan albumin protein, or a fragment or variant thereof) and/orheterologous control regions (e.g., promoter and/or enhancer) withendogenous polynucleotide sequences encoding a Therapeutic protein viahomologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issuedJun. 24, 1997; International Publication Number WO 96/29411;International Publication Number WO 94/12650; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989), the disclosures of each of which are incorporated byreference in their entireties).

Heteromultimer proteins described herein can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, hydrophobic charge interaction chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

In certain embodiments the heteromultimer proteins of the invention arepurified using Anion Exchange Chromatography including, but not limitedto, chromatography on Q-sepharose, DEAE sepharose, poros HQ, poros DEAF,Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE,Fractogel Q and DEAE columns.

In specific embodiments the proteins described herein are purified usingCation Exchange Chromatography including, but not limited to,SP-sepharose, CM sepharose, poros HS, poros CM, Toyopearl SP, ToyopearlCM, Resource/Source S and CM, Fractogel S and CM columns and theirequivalents and comparables.

In addition, heteromultimer proteins described herein can be chemicallysynthesized using techniques known in the art (e.g., see Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., N.Y and Hunkapiller et al., Nature, 310:105-111 (1984)). Forexample, a polypeptide corresponding to a fragment of a polypeptide canbe synthesized by use of a peptide synthesizer. Furthermore, if desired,nonclassical amino acids or chemical amino acid analogs can beintroduced as a substitution or addition into the polypeptide sequence.Non-classical amino acids include, but are not limited to, to theD-isomers of the common amino acids, 2,4diaminobutyric acid, alpha-aminoisobutyric acid, 4aminobutyric acid, Abu, 2-amino butyric acid, g-Abu,e-Ahx, 6amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

Provided are heteromultimers which are differentially modified during orafter translation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited, to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed herein include,for example, e.g., N-linked or O-linked carbohydrate chains, processingof N-terminal or C-terminal ends), attachment of chemical moieties tothe amino acid backbone, chemical modifications of N-linked or O-linkedcarbohydrate chains, and addition or deletion of an N-terminalmethionine residue as a result of procaryotic host cell expression. Theheteromultimer proteins are modified with a detectable label, such as anenzymatic, fluorescent, isotopic or affinity label to allow fordetection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include iodine, carbon,sulfur, tritium, indium, technetium, thallium, gallium, palladium,molybdenum, xenon, fluorine.

In specific embodiments, heteromultimer proteins or fragments orvariants thereof are attached to macrocyclic chelators that associatewith radiometal ions.

As mentioned, the heteromultimer described herein is modified by eithernatural processes, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given polypeptide.Polypeptides of the invention may be branched, for example, as a resultof ubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993);POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992)).

In certain embodiments, heteromultimeric proteins may also be attachedto solid supports, which are particularly useful for immunoassays orpurification of polypeptides that are bound by, that bind to, orassociate with albumin fusion proteins of the invention. Such solidsupports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

In embodiments where the heteromultimeric protein comprises only the VHdomain of an antibody, it may be necessary and/or desirable to coexpressthe protein with the VL domain of the same antibody, such that theVH-albumin fusion protein and VL protein will associate (eithercovalently or non-covalently) post-translationally.

In embodiments where the heteromultimeric protein comprises only the VLdomain of an antibody, it may be necessary and/or desirable to coexpressthe fusion protein with the VH domain of the same antibody, such thatthe VL-albumin fusion protein and VH protein will associate (eithercovalently or non-covalently) post-translationally.

Also provided herein are chemically modified derivatives of theheteromultimeric proteins which may provide additional advantages suchas increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337).The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The proteins may be modified at random positions withinthe molecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a Therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 105,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000,70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

The presence and quantity of heteromultimer proteins described hereinmay be determined using ELISA, a well known immunoassay known in theart. In one ELISA protocol that would be useful fordetecting/quantifying heteromultimers described herein, comprises thesteps of coating an ELISA plate with an anti-human serum albuminantibody, blocking the plate to prevent non-specific binding, washingthe ELISA plate, adding a solution containing the protein describedherein (at one or more different concentrations), adding a secondaryanti-cargo polypeptide specific antibody coupled to a detectable label(as described herein or otherwise known in the art), and detecting thepresence of the secondary antibody. In an alternate version of thisprotocol, the ELISA plate might be coated with the anti-cargopolypeptide specific antibody and the labeled secondary reagent might bethe anti-human albumin specific antibody.

Provided herein are multifunctional heteromultimers that comprise: atleast two monomers, wherein at least one monomer comprises at least onecargo molecule attached to a transporter polypeptide, such that saidmonomers associate to form the heteromultimer; wherein at least onetransporter polypeptide is derived from a monomeric protein and whereinsaid transporter polypeptides self-assemble to form a quasi-nativestructure of said monomeric protein or analog thereof. In certainembodiments, the cargo molecule is a biomolecule. In specificembodiments is a heteromultimer that comprises: at least two monomericproteins, wherein each monomeric protein comprises at least one cargopolypeptide, attached to a transporter polypeptide, such that saidmonomeric proteins self-assemble to form the heteromultimer. In certainembodiments, the heteromultimer is a heterodimer. In an embodiment, theheteromultimer is bispecific. In an embodiment, the heteromultimer ismultispecific. In certain embodiments, at least one transporterpolypeptide is not derived from an antibody. In certain embodiments, thetransporter polypeptides are not derived from an antibody. In anembodiment, the heteromultimer is multifunctional. In certainembodiments, the transporter polypeptides are derivatives of albumin. Incertain embodiments of the heteromultimer described herein, thetransporter polypeptides are derived from human serum albumin of SEQ IDNo. 1. In certain embodiments of the heteromultimer described herein,the transporter polypeptides are derived from alloalbumins. In certainembodiments, the cargo polypeptides are therapeutic proteins describedherein, or fragments or variants thereof. In some embodiments, at leastone cargo polypeptide is fused to the transporter polypeptide. Incertain embodiments, at least one cargo polypeptide is attached to theN-terminus of the transporter polypeptide. In some embodiments, at leastone cargo polypeptide is attached to the C-terminus of the transporterpolypeptide.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomer that comprises at least one cargo molecule, and afirst transporter polypeptide; and at least a second monomer thatcomprises at least one cargo molecule and a second transporterpolypeptide wherein at least one transporter polypeptide is derived froma monomeric protein and wherein said transporter polypeptidesself-assemble to form a quasi-native structure of said monomeric proteinor analog thereof. In certain embodiments, at least one cargo moleculeis a therapeutic agent described herein. In certain embodiments, atleast one cargo molecule is a biomolecule described herein. Providedherein are heteromultimers, each heteromultimer comprising: at least afirst monomeric protein that comprises at least one cargo polypeptideand a first transporter polypeptide; and at least a second monomericprotein that comprises at least one cargo polypeptide and a secondtransporter polypeptide. In certain embodiments, the heteromultimer is aheterodimer. In certain embodiments, the heteromultimer is multivalent.In an embodiment, the heteromultimer is bivalent. In some embodiments,the heteromultimer is multispecific. In an embodiment, theheteromultimer is bispecific. In certain embodiments, the transporterpolypeptides are derivatives of albumin. In certain embodiments of theheteromultimer described herein, the transporter polypeptides arederived from human serum albumin of SEQ ID No. 1.

In certain embodiments, are heteromultimers, each heteromultimercomprising: at least a first monomeric protein that comprises at leastone cargo polypeptide and a first transporter polypeptide comprising asequence of SEQ ID NO:2; and at least a second monomeric protein thatcomprises at least one cargo polypeptide and a second transporterpolypeptide comprising a sequence of SEQ ID NO: 3. In certainembodiments of the heteromultimer described herein, at least onetransporter polypeptide is derived from alloalbumins. In certainembodiments, both transporter polypeptides are derived fromalloalbumins. In certain embodiments, all transporter polypeptides arederivatives of the same alloalbumin. In some other embodiments, thetransporter polypeptides are derivatives of different alloalbumins. Insome embodiments, each transporter polypeptide is an alloalbuminderivative based on an alloalbumin selected from Table 2. In certainembodiments, the first monomeric protein comprises two cargopolypeptides. In some embodiments, the second monomeric proteincomprises two cargo polypeptides.

In some embodiments of the heteromultimer described herein, thetransporter polypeptides are derivatives of an annexin protein. In anembodiment, the transporter polypeptides are derived from differentannexin proteins. In certain embodiments, the transporter polypeptidesare derived from the same annexin protein. In an embodiment, at leastone transporter polypeptide is derived from Annexin A1 or lipocortin I.In certain embodiments of the heteromultimer, all transporterpolypeptides are derived from Annexin A1 of SEQ ID NO: 14. In certainembodiments of the heteromultimer, at least one transporter polypeptidesis derived from a sequence homologous to SEQ ID NO: 14. In anembodiment, at least one transporter polypeptide is derived from AnnexinA2 or annexin II. In certain embodiments of the heteromultimer, alltransporter polypeptides are derived from Annexin A2 or lipocortin II.In an embodiment, at least one transporter polypeptide is derived fromAnnexin like protein. In certain embodiments of the heteromultimer, alltransporter polypeptides are derived from Annexin like protein. In anembodiment, at least one transporter polypeptide is derived from thegroup comprising Annexin A1-Annexin A7. In an embodiment of theheteromultimer described herein, all transporter polypeptides arederived from the group comprising Annexin A1-Annexin A7. 14. In certainembodiments, the first annexin based transporter polypeptide has asequence comprising SEQ ID NO:15, and the second annexin basedtransporter polypeptide has a sequence comprising SEQ ID NO: 16.

In some embodiments of the heteromultimer described herein, thetransporter polypeptides are derivatives of transferrin. In anembodiment, at least one transporter polypeptide is derived fromtransferrin. In certain embodiments of the heteromultimer, at least onetransporter polypeptides are derived from transferrin of SEQ ID NO: 19or analog thereof. In certain embodiments of the heteromultimer, atleast one transporter polypeptide is derived from a polypeptide sequencehomologous to the transferrin. In certain embodiments of theheteromultimer described herein, at least one transporter polypeptide isderived from apo-transferrin. In certain embodiments, the firsttransferrin based transporter polypeptide has a sequence comprising SEQID NO:15 and the second transferrin based transporter polypeptide has asequence comprising SEQ ID NO: 16. Provided herein are heteromultimers,each heteromultimer comprising: at least a first monomeric protein thatcomprises at least one cargo polypeptide and a first transporterpolypeptide; and at least a second monomeric protein that comprises atleast one cargo polypeptide and a second transporter polypeptide,wherein said cargo polypeptides are selected from the proteins listed inTable 2, and wherein at least one transporter polypeptide is derivedfrom a monomeric protein and wherein said transporter polypeptidesself-assemble to form a quasi-native structure of said monomeric proteinor analog thereof. In certain embodiments, are heteromultimers, eachheteromultimer comprising: at least a first monomeric protein thatcomprises at least one cargo polypeptide and a first transporterpolypeptide; and at least a second monomeric protein that comprises atleast one cargo polypeptide and a second transporter polypeptide,wherein at least one at least one cargo polypeptide is an antibody, orfragment or variant thereof. In certain embodiments, all cargopolypeptides are antibodies or fragments or variants thereof. In certainembodiments, at least one cargo molecule attached to the firsttransporter polypeptide is the same as at least one cargo moleculeattached to the second transporter polypeptide. In certain embodiments,the cargo molecules attached to the first transporter polypeptide aredifferent from the cargo molecule on the second transporter polypeptide.In certain embodiments, there are at least two cargo molecules attachedto the first transporter polypeptide and at least two cargo moleculeattached to the second transporter polypeptide. In certain embodimentsthe cargo molecules attached to the first transporter polypeptide arethe same. In certain embodiments at least two cargo molecules attachedto the first transporter polypeptide are different from each other. Incertain embodiments at least two cargo molecules attached to the secondtransporter polypeptide are the same. In certain embodiments at leasttwo cargo molecules attached to the second transporter polypeptide aredifferent. In some embodiments, the antibody fragment comprises antibodyFc region. In some embodiments, the antibody is an immunoglobulinselected from the group consisting of IgG, IgA, IgD, IgE, and IgM. Incertain embodiments, the IgG is of subtype selected from IgG1, IgG2a,IgG2b, IgG3 and IgG4. In certain embodiments, the antibody is amultispecific antibody. In some embodiments, the multispecific antibodyis a bispecific antibody.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein at least one cargo polypeptideis a therapeutic antibody. In some embodiments of the heteromultimersdescribed herein, at least one cargo polypeptide is a therapeuticantibody or fragment or variant thereof, wherein the antibody isselected from antibody is selected from abagovomab, adalimumab,alemtuzumab, aurograb, bapineuzumab, basiliximab, belimumab,bevacizumab, briakinumab, canakinumab, catumaxomab, certolizumab pegol,certuximab, daclizumab, denosumab, efalizumab, galiximab, gemtuzumabozagamicin, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab,lumiliximab, mepolizumab, motavizumab, muromonab, mycograb, natalizumab,nimotuzumab, ocrelizumab, ofatumumab, omalizumab, palivizumab,panitumumab, pertuzumab, ranizumab, reslizumab, rituximab, teplizumab,toclizumab, tositumomab, trastuzumab, Proxinium, Rencarex, ustekinumab,and zalutumumab. In certain embodiments, the therapeutic antibody bindsa cancer antigen.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein at least one cargo polypeptideis an enzyme, hormone, therapeutic polypeptide, antigen, chemotoxin,radiotoxin, cytokine or variant or fragment thereof.

Provided herein are heteromultimers, each heteromultimer comprising: atleast a first monomeric protein that comprises at least one cargopolypeptide and a first transporter polypeptide; and at least a secondmonomeric protein that comprises at least one cargo polypeptide and asecond transporter polypeptide, wherein the cargo polypeptide isattached to the transporter polypeptide by chemical conjugation, nativeligation, chemical ligation, a disulfide bond or fusion.

Provided herein are host cells comprising nucleic acid encoding aheteromultimer described herein. In certain embodiments, the nucleicacid encoding the first monomeric protein and the nucleic acid encodingthe second monomeric protein are present in a single vector. In certainembodiments, the nucleic acid encoding the first monomeric protein andthe nucleic acid encoding the second monomeric protein are present inseparate vectors.

Provided herein is a method of making a heteromultimer, wherein saidmethod comprises: culturing a host cell described herein such that thenucleic acid encoding a heteromultimer described herein is expressed;and recovering the heteromultimer from the cell culture. In someembodiments, the host cell is a prokaryotic cell or a eukaryotic cell.In certain embodiments, the host cell is yeast cell. In someembodiments, the yeast is S. cerevisiae. In some embodiments, the yeastis glycosylation deficient, and/or protease deficient. In someembodiments, the host cell is a bacterial cell. In some embodiments, thehost cell expressing a heteromultimer descried herein is a mammaliancell. In certain embodiments, the mammalian cell is a CHO cell, a BHKcell, NSO cell, COS cell or a human cell.

Provided is a pharmaceutical composition that comprises a heteromultimerdescribed herein and a pharmaceutically acceptable adjuvant. Alsoprovided are methods of treating an individual suffering from a diseaseor disorder, said method comprising administering to the individual aneffective amount of a formulation or pharmaceutical compositiondescribed herein. In certain embodiments is a method of treating cancerin a patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In some embodiments is a method of treating an immune disorder in apatient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.Also provided is a method of treating an infectious disease in apatient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating a cardiovascular disorderin a patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.In certain embodiments is a method of treating a respiratory disorder ina patient, said method comprising administering to the patient atherapeutically effective amount of a heteromultimer described herein.

Provided is a kit for detecting the presence of a biomarker of interestin an individual, said kit comprising (a) an amount of a heteromultimerdescribed herein, wherein said heteromultimer comprises at least onecargo polypeptide such that said cargo polypeptide is capable of bindingto the biomarker of interest; and (b) instructions for use.

Provided herein are heteromultimer proteins that comprise at least twomonomeric proteins, wherein each monomeric protein comprises at leastone cargo polypeptide, and an albumin based polypeptide, such that saidmonomeric proteins self-assemble to form the heteromultimer.

In certain embodiments, the cargo polypeptide is fused to the albumin oralloalbumin based polypeptide. In some embodiments, the cargopolypeptide is chemically conjugated to the albumin or alloalbumin basedpolypeptide. In certain embodiments, the cargo polypeptide is attachedto the albumin or alloalbumin based polypeptide by means of chemicalligation or a disulfide bond.

Provided herein are heteromultimer proteins that comprise at least twomonomeric proteins, wherein each monomeric protein comprises at leastone cargo polypeptide, and an alloalbumin based polypeptide, such thatsaid alloalbumin based polypeptides self-assemble to form theheteromultimer with a quasi-native structure of said alloalbumin oranalog thereof. In some embodiments, a heteromultimer described hereinis a heterodimer. In some embodiments cargo polypeptide is an antibody,enzyme, hormone, therapeutic polypeptide, antigen, chemotoxin,radiotoxin, cytokine or variant or fragment thereof. In someembodiments, the cargo polypeptide of one monomeric protein functions insynergy with the cargo polypeptide of another monomeric protein.

In an aspect described herein is a method to derive protein segmentsfrom a protein of interest that can efficiently fold and selectivelyassociate together to form an active quasi-native protein likestructure.

Provided herein is a strategy for creating polypeptides based on amonomeric protein such as but not restricted to human serum albumin(HSA) that yield a quasi-native monomeric protein like structure andfunction when associated with each other. In embodiments describedherein, this strategy is also used to design heteromultimers comprisingmonomeric polypeptides that comprise transporter polypeptides that arederivatives of HSA variants, alloalbumins other homologous albuminmolecules from other species and also Annexin and Transferrin. Themonomers described herein can be engineered using a variety ofstrategies to improve biophysical characteristics such as the stabilityof the individual transporter polypeptides or their associated complex.

In an embodiment is a scaffold for the development of bispecific orother multispecific or multifunctional protein molecules based onfragments derived from HSA.

Provided is a transporter polypeptide which is a HAS, HAA, Annexin orTransferrin derived scaffold that can be conjugated or fused with cargopolypeptides such as other functional domains such as antigen bindingprotein units, target substrates or inhibitors or payloads such aschemotoxins, radiotoxins, cytokines, etc. to achieve a multispecific ormultifunctional therapeutic protein.

Described herein are fusions of heterodimeric Fc with transporterpolypeptides based on HSA to yield bispecific antibody basedtherapeutics with sufficient purity and stability for pharmaceuticalapplications.

In an aspect, described herein is a method of deriving a multispecificor multifunctional protein comprising self-assembling monomers thatcomprise transporter polypeptides based on HSA, such that, the proteinhas a number of favorable pharmacokinetic properties including improvedhalf-life, improved stability, low immunogenicity, etc.

Provided herein are heterodimer proteins that comprise at least twomonomeric fusion proteins, wherein each monomeric fusion proteinscomprises at least one cargo polypeptide fused to an albumin derivedpolypeptide, such that said albumin derived polypeptides self-assembleto form the multifunctional heterodimer with a quasi-native structure ofalbumin or an analog thereof.

In certain embodiments are heterodimer proteins that comprise at leasttwo monomeric fusion proteins, wherein each monomeric fusion proteinscomprises at least one cargo polypeptide fused to an alloalbumin derivedpolypeptide, such that said alloalbumin derived polypeptidesself-assemble to form the multifunctional heterodimer.

In certain embodiments described herein are heteromultimer proteins thatcomprise at least two monomeric fusion proteins, wherein each monomericfusion proteins comprises at least one cargo polypeptide fused to analloalbumin derived polypeptide, such that said alloalbumin derivedpolypeptides self-assemble to form the multifunctional heterodimer. Incertain embodiments are heterodimeric proteins comprising a firstmonomer which comprises at least one cargo polypeptide fused to analloalbumin derived polypeptide; and a second monomer that comprises atleast one cargo polypeptide fused to an alloalbumin derived polypeptide.In certain embodiments, the at least one cargo polypeptide of the firstmonomer is different from the at least one cargo polypeptide of thesecond monomer.

Provided herein is a heteromultimer that comprises: at least twomonomers, each comprising a transporter polypeptide and optionally atleast one cargo molecule attached to said transporter polypeptide,wherein each transporter polypeptide is obtained by segmentation of awhole protein such that said transporter polypeptides self-assemble toform quasi-native whole protein. In certain embodiments, theheteromultimer is multispecific. In certain embodiments, the transporterpolypeptides are not derived from an antibody. In some embodiments, eachmonomer preferentially forms the heteromultimer as compared to a monomeror a homomultimer. In an embodiment of the heteromultimer, at least onecargo molecule is a therapeutic agent, or a biomolecule. In someembodiments, at least one cargo molecule is a biomolecule which isselected from a polypeptide, DNA, PNA, or RNA. In some embodiments, eachtransporter polypeptide is a derivate of albumin or alloalbumin. In anembodiment, each transporter polypeptide is a derivate of annexin. Incertain embodiments, each transporter polypeptide is a derivate oftransferrin.

In certain embodiments are pharmaceutical formulations that comprise analbumin-based and/or alloalbumin-based heteromultimeric proteindescribed herein and a pharmaceutically acceptable diluent or carrier.In certain embodiments, a formulation described herein is provided aspart of a kit or container. In certain embodiments, the kit or containeris packaged with instructions pertaining to extended shelf life of thetherapeutic protein. In some embodiments, a heteromultimer describedherein is used in a method of treating (e.g., ameliorating) preventing,or diagnosing a disease or disease symptom in an individual, comprisingthe step of administering said formulation to the individual.

Also provided are transgenic organisms modified to contain nucleic acidmolecules described herein to encode and express monomeric fusionproteins described herein.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES Example 1 The Protein Splitting Method

Specific protein-protein association is driven by strong surfacecomplementarity between interacting partners and the accompanyingstructural and thermodynamic changes. The surface complementarityprovides an opportunity to form contacts that support the creation offavorable electrostatic and hydrophobic interactions. Electrostaticinteractions involve the formation of salt bridges, hydrogen bonds andthe pervasive dispersion interactions. Solvent exclusion andreorganization around non-polar atomic groups at the interface and itsassociated entropic effects play a role in the hydrophobic component ofthe binding thermodynamics. Residues with geometries that are optimizedfor hydrophobic interaction with one another will form contacts (i.e.stacking, pi-pi, cation-pi contacts favorable for stabilizing aprotein-protein interface. Similar thermodynamic effects controlmulti-step protein folding processes that involve the pre-organizationof secondary structural units and tertiary domains, which is followed bytheir association to form the folded quaternary state of the protein. Analternate mechanism to protein folding and binding involves a coupledprotein folding and binding process that ultimately results in thequaternary state of the protein. In the context of protein association,the individual protein components need to be co-expressed or be presentin the same medium and each of the components or monomers will stablyfold into its final structural state only on association with itsobligate partner. (FIG. 6)

Generation of a split protein involves recognizing a segmentation sitein the native protein, using information from sequence, secondarystructure and fold that will yield at least two transporter polypeptidesthat efficiently form the quasi-native protein structure byself-assembling to form a heteromultimer together. For example, thesesplit protein transporter polypeptides selectively self-assemble andform the quasi-native state when co-expressed. While generating a splitprotein complementary pair of transporter polypeptides, in a way, theattempt is to emulate a number of naturally occurring obligateprotein-protein complexes that exhibit their functionality as a complexwhile being non-functional in their uncomplexed state. A successfulimplementation of the strategy results in polypeptides that selectivelyself-assemble to form heteromultimers with each other, are soluble asindividual entities and for functional relevance, do not impair thefolding, binding and activity of other components in the environment.The intrinsic nature of the polypeptides to reconstitute with each otherhas applications in area of creating heteromultimeric fusion entitiesout of cargo molecules that are not efficient at forming multimers bythemselves. The functional role of the split protein segments is to actas transporter polypeptides that drive heteromultimerization.

Example 2 Preparation of HA/Alloalbumin Based Heteromultimer Proteins

Shown is a method to determine the segmentation site along the HSAsequence and structure that will yield monomeric polypeptide chains thatstably fold and fuse to form a quasi-native quaternary structure of theoriginal protein. One of the critical requirements for such stableassociation is the formation of a large buried area of surfacecomplementarity at the interface between the polypeptide chains. Thenative fold of the original protein provides indication of the naturalcomplementarity of regions within the protein.

FIG. 2 shows the solvent accessible surface area buried at the interfaceof two albumin-based polypeptides that would ideally fold into thequasi-native structure of HSA, when the segmentation point is movedalong the protein sequence. The analysis indicates that a large surfacearea, of the order of about 2000 Å² is buried when the splitsegmentation is introduced anywhere between residues 30 and 520 with afew exceptions. Albumin has an exceptionally large number of disulphidesbridges that contributes to the stability of the native proteinstructure. Section of the protein near residues 110, 190, 300, 390 and500 provide sites for segmentation that do not split the residuesinvolved in a disulphide link across the two transporter polypeptides.Segmentation in other regions would result in heterodimers with a crosslinking disulphide bond between the two transporter polypeptide pairs.FIG. 3 presents a model representation of one such quasi-native albuminstructure derived by removal of loop from residues 294 to 303 in the HSAsequence. The total buried surface area for the two albumin basedpolypeptides of SEQ ID No. 2, and SEQ ID No: 3 shown herein isapproximately 2500 Å². This is within the average range of 1910-3880 Å²observed in a number of protein-protein heterodimeric and homodimericco-complex structures [Bahadhur R. P. & Zacharias M. (2008) Cell MolLife Sci 65, 1059-1072]. This suggests that there is a strong likelihoodfor the two polypeptides to selectively associate with each other if thefolding pathway of the two polypeptides is fairly independent of eachother.

In an aspect of this invention, selective formation of a stablequasi-native structure with the two polypeptides (the pair formed by SEQID No. 2 and SEQ ID No. 3 or the transporter pair formed by SEQ ID No. 8and SEQ ID No. 10) gives us the opportunity to employ these polypeptidesto drive the formation of bispecific or other multifunctional moleculesafter fusing the appropriate cargo proteins of interest to the N or Cterminus of the albumin based polypeptides employed as transporterpolypeptides. A number of other alternate segmentation patternsresulting in transportation polypeptide pair heterodimer can bedesigned. The fused cargo proteins can be antigen binding domains orother payloads such as chemotoxins, radiotoxins or cytokines (asrepresented in FIG. 4). The resulting heterodimers have many of thefavorable properties intrinsic to HSA including properties like improvedhalf-life, stability and low immunogenicity. Traditional linkers such as(Gly₄Ser)_(x) can be used for the association of the cargo protein withthe transporter polypeptide.

In another aspect of this invention, each of the HSA based transporterpolypeptides is fused independently to the C-terminus of two heavychains in a bispecific Fc molecule (as represented in FIG. 5). Thestrong and selective pairing of the two transporter polypeptides (suchas SEQ ID No. 2, and SEQ ID No. 3) drives the selectivelyheterodimerization of the Fc and also contribute to its stability andother valuable pharmacokinetic properties.

Serum albumin preprotein NP_000468.1 GI 4502027 mRNA sequence fromNM_000477.5, Consensus CDS (CCDS) ID 3555.1

SEQ ID No. 4: Residue 1-29 (EFATMAVMAPRTLVLLLSGALALTQTWAG) is theN-terminal export signal sequence region that gets cleaved. Thissequence fulfills the same role as the natural signal sequence but it'soptimized for mammalian and CHO cell lines.

SEQ ID No. 1: gi|4502027|ref|NP_000468.1|serum albumin preproprotein [Homo sapiens]EFATMAVMAPRTLVLLLSGALALTQTWAGDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAAL GLSEQ ID No. 5: Human serum albumin nucleotide CCDS Sequence (1852 nt)    GAATTCGCCACTATGGCTGTGATGGCCCCTAGGACCCTGGTGCTGCTGCTGTCCGGAGCTCTGGCTCTGACTCAGACCTGGGCTGGAGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGC CTTAGGCTTATGA

The protein and nucleotide sequence of albumin based polypeptides usefulas transporter polypeptides are as follows:

Albumin based heteromultimer 1:Albumin based Transporter polypeptide 1-Ver 1: SEQ ID No. 2:    DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVNucleotide sequence encoding Albumin basedTransporter polypeptide 1-Ver 1: SEQ ID No. 6:    GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCA CTGCATTGCCGAAGTGTGAAlbumin based Transporter polypeptide 2-Ver 1: SEQ ID No. 3:    SLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLSKCCAAADPHECYAKVFBSFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLKEKTPVSDRVTKCCTESLYNRRPCFSALEVDETYVPKEFNAETFTFKADICTLSEKERQIKXQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLNucleotide sequence encoding Albumin based Transporter polypeptide 2-Ver 1: SEQ ID No. 7:    TCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATGA Albumin based heteroMultimer 2:Albumin based Transporter polypeptide 1-Ver 2: SEQ ID No. 8:    DAHKSEVAHRFKDLGSENFKALVLIAFAQYLQQCPFHDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQSPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYSIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDSGKASSAKQRLKCASLQKFGERAFKaWAVARLSQRFPKAEFAEVSKLVTBLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLSKSHCIAEVSNDEMPABLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARANucleotide sequence encoding Albumin basedTransporter polypeptide 1-Ver 2: SEQ ID No. 9:    GATGCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGGTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAGCATGAAlbumin based Transporter polypeptide 2-Ver 2: SEQ ID No. 10:    SVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEBPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSECCKHPEAKRMPCAEDYLSVVLKQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLNucleotide sequence encoding Albumin basedTransporter polypeptide 2-Ver 2: SEQ ID No. 11:    TCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGC

Generation and Expression of HA or HAA Based Heteromultimers

The genes encoding the full length WT HA and the HA based transporterpolypeptide monomers were constructed via gene synthesis using codonsoptimized for human/mammalian expression. The constructs were designedfrom known full-length Human Serum Albumin Preprotein (GENEBANK:NP_000468.1), after exclusion of the signal sequenceEFATMAVMAPRTLVLLLSGALALTQTWAG. The final gene products were subclonedinto the mammalian expression vector pTT5 (NRC-BRI, Canada) (Durocher etal). High level and high-throughput recombinant protein production bytransient transfection of suspension-growing human CHO-3E7 wasperformed. See Table 3 for construct boundaries of the two scaffoldsdescribed here: Albumin based heteromultimer 1 (ABH1) and Albumin basedheteromultimer 2 (ABH2). Albumin based heteromultimer 2 comprises onedisulfide bond between the two transporter polypeptides, while Albuminbased heteromultimer 1 is formed entirely by non-covalent interactions.FIG. 6A provides SDS-PAGE (non-reducing) gel analysis of the twoheteromultimer constructs (ABH1 and ABH2), after co-expression(different DNA transfection ratios are shown). WT full-length HSA isshown as control. As expected, ABH2 retains the disulfide linkage innon-reducing SDS-PAGE, with a MW roughly double the non-disulfide linkedABH1. FIG. 6B provides Native gel analysis of the two Albumin basedheteromultimer constructs (ABH1 and ABH2), after co-expression (1:1 DNAlevel). WT full-length HSA is shown as control. ABH1 and ABH2 both forma complex of expected mass, comparable to the full-length WT HSA.Furthermore, upon expression, neither the transporter polypeptidesforming ABH1 nor the ones forming ABH2 homodimerize; rather theypreferably form a stable hetercomplex. See Table 3 below for details.

TABLE 3 Albumin based heteromultimer constructs MW Construct SegmentBoundaries* (KDa) Wild Type HA 1:585 (SEQ ID NO: 1) 64.3 ABH1 1:293 (SEQID NO: 2) 32.2 304:585 (SEQ ID NO: 3)  30.9 ABH2 1:337 (SEQ ID NO: 8) 37342:585 (SEQ ID NO: 10)  26.7

WT-HSA and the two Albumin based heteromultimers (ABH1 and ABH2) wereexpressed in CHO-3E7 cell line grown in suspension in FreeStyle F17medium (Invitrogen) supplemented with 0.1% w/v pluronic and 4 mMglutamine. The day of transfection cell density should be around 1.5-2million cells/ml and viability must be greater than 97%. Transfection isdone according to patent application WO 2009/137911 using a mixture ofplasmid DNA made of 5% pTTo-GFP plasmid (green fluorescent protein todetermine transfection efficiency, Table 4), 15% pTT22-AKT plasmid, 21%HSA plasmids (10.63% of each), 68.37% of Salmon Sperm DNA. Followingtransfection, the shake flask containing cells is then placed on anorbital shaker set to 120 rpm in a humidified incubator with 5% CO2 at37° C. Twenty-four hours post-transfection, 1% w/v TN1 and 0.5 mM VPA(Valproic acid) are added to the cultures. The cultures are thentransferred on an orbital shaker (120 rpm) placed in a humidifiedincubator with 5% CO2 set at 32° C. At 24-48 hours, GFP positive cellsshould be between 30-60% as determined by flow cytometry. Cells wereharvested 7 days post-transfection and spun at 4,000 rpm for 20 minutes.The supernatant was filter-sterilized (clarified) using a 0.45 μm filter(Millipore). Keep the supernatant at 4° C. for short period storage andat −80° C. for long period storage. Prior to purification, the frozensupernatant was thawed at 37° C., re-filtered and degassed through a0.45 μm membrane filter under vacuum for 5-10 minutes.

TABLE 4 Cell viability at different stages of expression for WT and ABH1construct. HSA % GFP 48 hrs % viability 48 hrs % viability 48 hrsscaffold post-transfection post-transfection post-transfection Wild Type67 94.6 72.3 HSA ABH2 66.3 93.6 77.1

Purification of HSA and Heteromultimers ABH1 and ABH2

Purification was performed by gravity flow using a bench-top QIAGEN-tip500 column packed with a Blue Sepharose matrix (GE Healthcare). The BlueSepharose matrix was equilibrated with 20 ml of PBS pH 7.2. The samplewas loaded at a flow rate of 5 ml/min and subsequently washed with 20 mlof PBS. The protein was eluted with 0.1 M Na2HPO4 pH 7.2 supplementedwith 1 M NaCl and collected in 1 ml fractions (20 ml total). Fractionscontaining HSA (as per Bradford protein assay) were pooled, and appliedon a HiLoad 16/60 Superdex 200 prep grade gel filtration column coupledto an AKTA Express system (GE Healthcare) using a flow rate of 1 ml/ml.Protein with a purity of >85% was collected; fractions containing puresample were pooled and concentrated by centrifugation using an AmiconUltra membrane with a cutoff weight of 10 000 MWCO. FIG. 6C showsSDS-PAGE (non-reducing) analysis of the ABH2 heteromultimer and WT HSA,both after the final stage of purification. Both constructs show theexpected MW.

Stability Determination of Albumin Based Heteromultimers UsingDifferential Scanning Calorimetry (DSC)

All DSC experiments were carried out using a GE or MicroCal VP-Capillaryinstrument. The proteins were buffer-exchanged into PBS (pH 7.4) anddiluted to 0.3 to 0.7 mg/mL with 0.137 mL loaded into the sample celland measured with a scan rate of 1° C./min from 20 to 100° C. Data wasanalyzed using the Origin software (GE Healthcare) with the PBS bufferbackground subtracted. See Table 5 and FIG. 7 for resulting meltingtemperature determined.

TABLE 5 Melting temperature for Albumin based heteromultimers MeasuredMass Theoretical MW Tm Molecule (Da) (Da) ° C. HSA Wild 66620 66470 75Type ABH2 66100 65880 63

Evaluation of FcRn Binding of HSA and ABH2 Using Surface PlasmonResonance

As seen in FIGS. 8A-B, when HSA and a HSA-based heteromultimer areimmobilized on the SPR surface, affinity towards FcRn appears to becomparable between the full length WT HSA and ABH2, indicating FcRnbinding functionality of albumin is retained by the heteromultimerformed by the self-assembly of albumin based transporter polypeptides.The following Table 6 illustrates FcRn binding data. Values inparenthesis refer to standard deviation.

TABLE 6 Kinetic and Equilibrium fit of FcRn Binding of HSA and ABH2using Surface Plasmon Resonance Ka (1/Ms) Kd (1/s) KD (M) Grouped FittedGrouped Fitted Grouped Fitted HAS 5.3E+04 (7E+03) 7.0E−02 (2.0E−02)1.4E−06 (6.0E−07) Kinetic fit ABH2 5.0E+04 (4E+03) 4.2E−02 (8.0E−03)8.0E−07 (2.0E−07) Kinetic fit HAS 9.0E−07 (1.0E−07) Equilibrium Fit ABH29.0E−07 (1.0E−07) Equilibrium Fit

Example 3 Generation and Expression of Albumin Based Heteromultimerswith Mono- and Tetravalency Comprising Anti-Her2/Neu and Anti-CD16 scFvBioactive Fusions

Multivalent heteromultimer ABH2 was generated by expressing its singlemonomeric transporter polypeptides, SEQ ID NO: 8 and SEQ ID NO: 10,fused at one or both termini to cargo polypeptides that are eitherantiHer2scFv (4D5) and/or anti-CD16 scFv (NM3E). These form a set of 8base construct monomers based off transporter polypeptide 1 and 8 baseconstruct monomers based off transporter polypeptide 2. Differentcombinations of these base constructs were combined upon co-expressionto form heteromultimers displaying all combination of the two cargopolypeptides at any of the four terminal positions of the twotransporter polypeptides, ranging from monovalent to tetravalent.

As shown in FIG. 9, the bioactive cargo polypeptides were fused to theheteromultimer transporter polypeptides via a GGSG linker, for the Nterminus of one monomer and a longer (GGS)4GG linker for all othertermini in the other monomer.

Table 7 illustrates the 16 base constructs (Base construct #1-Baseconstruct #16) that were generated by fusing the 4D5 and NM3 cargopolypeptides to either N or C terminus of transporter polypeptide 1 (F1)or transporter polypeptide 2 (F2). F1: corresponds to SEQ ID 8 and F2corresponds to SEQ ID 10.

Single fusions # Fusion 1 Fusion 2 1 NM3E2 F1 2 F1 NM3E2 3 NM3E2 F2 4 F2NM3E2 5 4D5 F1 6 F1 4D5 7 4D5 F2 8 F2 4D5 Double fusions # Fusion 1Fusion 2 Fusion 3 9 NM3E2 F1 NM3E2 10 NM3E2 F2 NM3E2 11 4D5 F1 4D5 124D5 F2 4D5 13 NM3E2 F1 4D5 14 4D5 F1 NM3E2 15 NM3E2 F2 4D5 16 4D5 F2NM3E2

Multivalent constructs were generated as outlined in Example 2 usingheteromultimer ABH2. The final gene products were subcloned into themammalian expression vector pTT5 (NRC-BRI, Canada) (Durocher et al).High level and high-throughput recombinant protein production bytransient transfection of suspension-growing human CHO-3E7 wasperformed.

Purification was performed by application of the cellular supernatantwith expressed protein to a QIAGEN-tip 500 column packed with BlueSepharose matrix (GE Healthcare) coupled to an AKTA Express system (GEHealthcare) using a flow rate of 1 ml/ml. The column was equilibratedwith equilibrated with sample buffer composed of 20 ml of PBS pH 7.2,300 mM NaCl. The sample was loaded at a flow rate of 5 ml/min andsubsequently washed with sample buffer. The protein was eluted byapplication of NaCl gradient ranging from 300 mM to 2000 mM. Fractionseluting in higher salt concentration were the purest and were pooled,concentrated and subsequently applied to a HiLoad 16/60 Superdex 200prep grade gel filtration column coupled to an AKTA Express system (GEHealthcare) using a flow rate of 1 ml/ml. Protein with a purity of >85%was collected; fractions containing pure sample were pooled andconcentrated by centrifugation using an Amicon Ultra membrane with acutoff weight of 10 000 MWCO. FIGS. 10A-10B shows SDS-PAGE(non-reducing) analysis of the ABH2 heteromultimer fused to differentcargo polypeptides. The position of those polypeptides in theheteromultimer relative to the transporter polypeptides is outlined intable 8 below. All constructs showed the expected molecular weight.

TABLE 8 Monovalent, multivalent, and multispecific constructs that weregenerated by fusing the 4D5 and NM3 cargo polypeptides to either N or Cterminus of transporter polypeptide 1 or transporter polypeptide 2 ofABH2. N terminus- C terminus- N terminus- C terminus- transportertransporter transporter transporter polypeptide 1 polypeptide 1polypeptide 2 polypeptide 2 Variant (SEQ ID No: 8) (SEQ ID No: 8) (SEQID No: 10) (SEQ ID No: 10) Valency 513 NM3E monovalent 514 NM3Emonovalent 515 NM3E monovalent 516 NM3E monovalent 517 4D5 monovalent518 4D5 monovalent 519 4D5 monovalent 520 4D5 monovalent 521 NM3E NM3Ebivalent 522 NM3E NM3E bivalent 523 NM3E NM3E bivalent 524 NM3E NM3Ebivalent 525 4D5 4D5 bivalent 526 4D5 4D5 bivalent 527 4D5 4D5 bivalent528 4D5 4D5 bivalent 529 NM3E NM3E bivalent 530 NM3E NM3E bivalent 5314D5 4D5 bivalent 532 4D5 4D5 bivalent 543 NM3E 4D5 bispecific 544 NM3E4D5 bispecific 545 NM3E 4D5 bispecific 546 NM3E 4D5 bispecific 547 4D5NM3E bispecific 548 4D5 NM3E bispecific 549 4D5 NM3E bispecific 550 4D5NM3E bispecific 551 NM3E 4D5 bispecific 552 4D5 NM3E bispecific 553 NM3E4D5 bispecific 554 4D5 NM3E bispecific 593 4D5 NM3E bispecific 594 NM3E4D5 bispecific

SPR Binding of Monovalent ABH2 Fused to a Single antiCD16scFv

Purified heteromultimer ABH2 fused to a single antiCD16scFv to the Nterminus of transporter polypeptide SEQ ID 2 (construct v515) was usedin a binding experiment using Surface Plasmon Resonance (SPR). SolubleCD16 was covalently immobilized onto a CM5 surface and ABH2 fused toantiCD16scFv was captured and binding kinetics were determined.

SPR Supplies. GLM sensorchips, the Biorad ProteOn amine coupling kit(EDC, sNHS and ethanolamine), and 10 mM sodium acetate buffers werepurchased from Bio-Rad Laboratories (Canada) Ltd. (Mississauga, ON).Recombinant Her-2 protein was purchased from eBioscience (San Diego,Calif.). HEPES buffer, EDTA, and NaCl were purchased from Sigma-Aldrich(Oakville, ON). 10% Tween 20 solution was purchased from Teknova(Hollister, Calif.).

SPR Biosensor Assays. All surface plasmon resonance assays were carriedout using a BioRad ProteOn XPR36 instrument (Bio-Rad Laboratories(Canada) Ltd. (Mississauga, ON)) with HBST running buffer (10 mM HEPES,150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween 20 pH 7.4) at a temperature of25° C. The CD16 capture surface was generated using a GLM sensorchipactivated by a 1:5 dilution of the standard BioRad sNHS/EDC solutionsinjected for 300 s at 30 μL/min in the analyte (horizontal) direction.Immediately after the activation, a 4.0 μg/mL solution of CD16 in 10 mMNaOAc pH 4.5 was injected in the ligand (vertical) direction at a flowrate of 25 pt/min until approximately 3000 resonance units (RUs) wereimmobilized. Remaining active groups were quenched by a 300 s injectionof 1M ethanolamine at 30 μL/min in the analyte direction, and this alsoensures mock-activated interspots are created for blank referencing.

A 500 nM 3-fold dilution series of V515 was injected over 3000 RUsCD16aWT (L6) compared to blank (L5). Flow rate 50 uL/min for 120 s, witha 240 s disassociation phase. Injections were repeated in standardrunning buffer (DPBS/3.4 mM EDTA/0.05% Tween20) and running buffer withan additional 350 mM NaCl. Sensorgrams were aligned anddouble-referenced using the buffer blank injection and interspots, andthe resulting sensorgrams were analyzed using ProteOn Manager softwarev3.0. Typically, K_(D) values were determined from binding isothermsusing the Equilibrium Fit model. For high affinity interactions withslow off-rates, kinetic and affinity values were additionally determinedby fitting the referenced sensorgrams to the 1:1 Langmuir binding modelusing local R_(max), and affinity constants (K_(D) M) were derived fromthe resulting rate constants (k_(d) s⁻¹/k_(a) M⁻¹s⁻¹). All K_(D) valuesare reported as the mean and standard deviation from three independentruns.

As shown in Table 9, ABH2 heteromultimer fused to a single antiCD16scFvhas full activity and binds its target with good reproducibility and KDsimilar to the free anti CD16 scFv (NM3E).

TABLE 9 SPR data for monovalent ABH2 fused to a single antiCD16scFv.Injection #1 Injection #2 ka kd KD ka kd KD 1/Ms 1/s M 1/Ms 1/s M KD (M)Ave KD SD NM3E 5.37E+04 5.76E−03 1.07E−07 5.89E+04 6.03E−03 1.02E−071.05E−07 4.E−09 V515 Dec 8.11E+04 6.71E−03 1.10E−07 V515 Jan 5.56E+047.30E−03 1.31E−07

Example 4 Preparation of HA or HAA Based Heteromultimer Proteins WhereinCargo Protein(s) Comprise One or More EGF-A Like Domain.

The peptide sequence of the EGF-A domain in PCSK9 protein or anotherpolypeptide sequence homologous to the EGF-A domain, capable ofspecifically binding the low density lipoprotein receptor (LDL-R) isderived by sequencing or from a database such as GenBank. The cDNA forthe cargo polypeptide comprising EGF-A like domain is isolated by avariety of means including but not exclusively, from cDNA libraries, byRT-PCR and by PCR using a series of overlapping syntheticoligonucleotide primers, all using standard methods. In certainexamples, the cargo protein is engineered to improve stability, targetbinding features or other biophysical or therapeutically relevantproperties. The polypeptide is employed as the cargo protein in thecreation of a heteromultimer with application in the treatment ofhypercholesterolemia. The first and second monomeric fusion polypeptidesequence is derived by fusing the cargo protein sequence directly orwith an intermediate linker peptide to the N-terminus and/or C-terminusof HA or HAA based transporter polypeptide such as SEQ ID No: 2, SEQ IDNO: 3, SEQ ID NO: 8 or SEQ ID NO: 10. This monomeric fusion proteinsequence is reverse translated to its corresponding DNA sequence to beintroduced in an expression vector, sequence optimized for expression ina particular cell line of interest. The first and second monomericfusion proteins are transfected and coexpressed in the cell line ofinterest. In certain cases, the transfection is in 1:1 ratio for the twovectors. In some examples, the ratio is selected from 1.5:1, 2:1, 1:1.5,1:2 etc.

Example 5 Preparation of HA or HAA Based Heteromultimeric ProteinsWherein Cargo Protein(s) are the GLP-1 and/or Glucagon.

The peptide sequence of GLP-1 or another polypeptide sequence homologousto this peptide, capable of specifically binding the GLP-1 receptor oracting as a GLP-1 agonist is derived by sequencing or from a databasesuch as GenBank. Alternately, the peptide sequence of Glucagon oranother polypeptide sequence homologous to this peptide, capable ofspecifically binding the Glucagon receptor or acting as a Glucagonreceptor agonist is derived by sequencing or from a database such asGenBank. The cDNA for each cargo polypeptide comprising GLP-1 orGlucagon is isolated by a variety of means including but notexclusively, from cDNA libraries, by RT-PCR and by PCR using a series ofoverlapping synthetic oligonucleotide primers, all using standardmethods. In certain examples, these GLP-1 or Glucagon based cargopolypeptides are engineered to improve stability, target bindingfeatures or other biophysical or therapeutically relevant properties.These GLP-1 and Glucagon based polypeptides are employed as one or morecargo molecules in the creation of a heteromultimer with application inthe treatment of type-2 diabetes or another disease related to glucosemetabolism. The first and second monomeric fusion polypeptide sequenceis derived by fusing the cargo protein sequence directly or with anintermediate linker peptide to the N-terminus and/or C-terminus of HA orHAA based transporter polypeptide such as SEQ ID No: 2, SEQ ID NO: 3,SEQ ID NO: 8 or SEQ ID NO: 10. The fusion proteins can be monospecificwith either GLP-1 or Glucagon like polypeptides or be bispecific(coagonist) with both the GLP-1 and Glucagon like polypeptides. Eachmonomeric fusion protein sequence is reverse translated to itscorresponding DNA sequence to be introduced in an expression vector,sequence optimized for expression in a particular cell line of interest.The first and second monomeric fusion proteins are transfected andcoexpressed in the cell line of interest. In certain cases, thetransfection is in 1:1 ratio for the two vectors. In some examples, theratio is selected from 1.5:1, 2:1, 1:1.5, 1:2 etc

Sequence of Cargo molecule GLP-1 SEQ ID No: 12:HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG Sequence of Cargo molecule GlucagonSEQ ID NO: 13: HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

Example 6 Annexin Protein Repeat as Membrane-Sensing MultivalentScaffold

Annexin is split with an extensive interface to generate a multivalentheteromultimer scaffold comprising two transporter polypeptides. Annexinis a 346 residue protein (PDB ID 1MCX). Heteromultimer comprising twotransporter polypeptides based on annexin split in the region betweenresidue 186 and 194 is shown in FIG. 11. When co-expressed in solution,the large interfacial area between the two transporter polypeptidesleads to self-assembly of the heterodimer. The self-assembly of the twounits allows for the design of multivalent construct with transporterpolypeptides based on the annexin core. Two structures are available,Pig and Human. The two structures are superimposable with an rmsd of 0.6A. The following stretch of sequence can be removed from the humanAnnexin sequence DRSEDF (residues 160 through 165). The truncation doesnot break any secondary structure element and does not involveintroducing or removing any Proline residue.

Human annexin WT Sequence SEQ ID NO: 14:GSAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFRNALLSLAKGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN Sequence of Annexin based transporterpolypeptide-1: SEQ ID NO: 15:SAVSPYPTFNPSSDVAALHKAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSG DFRNALLSLAKGSequence of Annexin based transporter polypeptide-2: SEQ ID NO: 16:GVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKG DYEKILVALCGGN

FIG. 12 shows a plot of the buried solvent accessible surface area atthe interface of Annexin based transporter polypeptide-1 (ABT-1), andAnnexin based transporter polypeptide-2 (ABT-2). A split annexin nearresidue position 186 forms a heterodimer with about 3200 Å² of buriedsurface area. The transporter polypeptides such as ABT-1 and ABT-2 basedon Annexin can be used to attach cargo biomolecules using the samemethods as described above for albumin based transporter polypeptides.

Example 7 Transferrin as a Multivalent Scaffold

Based on the large number of therapeutically relevant properties oftransferrin, this protein presents itself as an interesting scaffoldmolecule for the design of multivalent protein fusion drugs followingthe creation of a self-assembling protein and its split component parts.The structure of transferrin is shown in FIG. 13 based on the crystalstructure (1H76) available in the protein data bank [Hall D R et al.Acta Crystallogr D 2002, 58, 70-80]. The transferrin molecule iscomposed of two structurally similar lobes, the N and C terminal lobes,connected by a short peptide linker between residues 333 and 342.

A heterodimer is designed based on transferrin protein, said heterodimercomprising a first transporter polypeptide involving residues 1-333 oftransferrin and a second transporter polypeptide composed of residuesfrom 342 to the C-terminus of the original transferrin sequence. Whencoexpressed, the two transporter polypeptides fold independently andpair to form a quasi-transferrin scaffold capable of maintaining itstherapeutically relevant properties. Furthermore, such a Transferrinscaffold allows for the production of multivalent fusion molecules, e.g.a multivalent GLP-1 fusion with transporter polypeptides based ontransferring. These fusions can be similar to the GLP-1-fusionpolypeptides with Albumin based transporter polypeptides.

FIG. 13 provides structure of transferrin molecule based on the PDBstructure 1H76. The two monomeric transporter polypeptides derived bysplitting the transferrin molecule are color coded as light and darkgrey units. The sites of fusion for the cargo molecules are representedas spheres. FIG. 14 shows a plot of the buried solvent accessiblesurface area at the interface of two transferrin based polypeptides. Asplit transferrin near residue position 330 such as the two transporterpolypeptides shown below, forms a heterodimer with about 1800 Å2 ofburied surface area.

Sequence of Transferrin based transporter polypeptide-1: SEQ ID NO: 17:MRLAVGALLV CAVLGLCLAV PDKTVRWCAV SEHEATKCQSFRDHMKSVIP SDGPSVACVK KASYLDCIRA IAANEADAVTLDAGLVYDAY LAPNNLKPVV AEFYGSKEDP QTFYYAVAVVKKDSGFQMNQ LRGKKSCHTG LGRSAGWNIP IGLLYCDLPEPRKPLEKAVA NFFSGSCAPC ADGTDFPQLC QLCPGCGCSTLNQYFGYSGA FKCLKDGAGD VAFVKHSTIF ENLANKADRDQYELLCLDNT RKPVDEYKDC HLAQVPSHTV VARSMGGKEDLIWELLNQAQ EHFGKDKSKE FQLFSSPHGK DLLFKDSAHGFLKVPPRMDA KMYLGYEYVT AIRNLREG.Sequence of Transferrin based transporter polypeptide-2: SEQ ID NO: 18:      ECKP VKWCALSHHE RLKCDEWSVN SVGKIECVSAETTEDCIAKI MNGEADAMSL DGGFVYIAGK CGLVPVLAENYNKSDNCEDT PEAGYFAVAV VKKSASDLTW DNLKGKKSCHTAVGRTAGWN IPMGLLYNKI NHCRFDEFFS EGCAPGSKKDSSLCKLCMGS GLNLCEPNNK EGYYGYTGAF RCLVEKGDVAFVKHQTVPQN TGGKNPDPWA KNLNEKDYEL LCLDGTRKPVEEYANCHLAR APNHAVVTRK DKEACVHKIL RQQQHLFGSNVTDCSGNFCL FRSETKDLLF RDDTVCLAKL HDRNTYEKYLGEEYVKAVGN LRKCSTSSLL EACTFRRP.

Example 9 Multiple Cargo Proteins

The heteromultimer proteins described herein (e.g, containing a cargopolypeptide (or fragment or variant thereof) fused to transporteralbumin segment or variant thereof) may additionally be fused to otherproteins to generate “multifusion proteins”. These multifusion proteinscan be used for a variety of applications. For example, fusion of theproteins described herein to His-tag IgG domains, and maltose bindingprotein facilitates purification. (See e.g EP A 394,827; Traunecker etal., Nature 331:84-86 (1988)). Nuclear localization signals fused to thepolypeptides can target the protein to a specific subcellularlocalization. Furthermore, the fusion of additional protein sequences toproteins described herein may further increase the solubility and/orstability of the heteromultimer. The heteromultimer proteins describedabove can be made using or routinely modifying techniques known in theart and/or by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian or yeast expression vector.

For example, if pC4 (ATCC Accession No. 209646) is used, the human Fcportion can be ligated into the BamHI cloning site. Note that the 3′BamHI site should be destroyed. Next, the vector containing the human Fcportion is re-restricted with BamHI, linearizing the vector, and apolynucleotide encoding a heteromultimeric protein described herein(generated and isolated using techniques known in the art), is ligatedinto this BamHI site. Note that the polynucleotide encoding the fusionprotein of the invention is cloned without a stop codon; otherwise an Fccontaining fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce theheteromultimeric protein described herein, pC4 does not need a secondsignal peptide. Alternatively, if the naturally occurring signalsequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., International Publication No.WO 96/34891.)

What is claimed:
 1. One or a combination of two or more nucleic acidmolecules encoding a heteromultimer, the heteromultimer comprising: afirst protein that comprises (i) a first transporter polypeptidecomprising a first segment of albumin; and (ii) at least one cargopolypeptide and a second protein that comprises (iii) a secondtransporter polypeptide comprising a second segment of albumin; and (iv)at least one cargo polypeptide, wherein said first segment of albuminand said second segment of albumin are derived from an albumin bysegmentation of the albumin, said first transporter polypeptide isdifferent from said second transporter polypeptide, and said transporterpolypeptides self-assemble to form a quasi-native albumin structure. 2.The nucleic acid molecules according to claim 1, wherein saidheteromultimer is a heterodimer.
 3. The nucleic acid molecules accordingto claim 2, wherein the first segment of albumin and the second segmentof albumin form a complementary pair of transporter polypeptides.
 4. Thenucleic acid molecules according to claim 1, wherein the first segmentof albumin and said second segment of albumin are derived from analbumin by segmentation of the albumin to remove a loop.
 5. The nucleicacid molecules according to claim 1, wherein said first transporterpolypeptide and said second transporter polypeptide are derived from amammalian albumin.
 6. The nucleic acid molecules according to claim 5,wherein the first transporter polypeptide and the second transporterpolypeptide are derived from the same type of albumin.
 7. The onenucleic acid molecules according to claim 6, wherein said firsttransporter polypeptide and said second transporter polypeptide arederived from the same alloalbumin.
 8. The nucleic acid moleculesaccording to claim 5, wherein the transporter polypeptides are derivedfrom different albumins.
 9. The nucleic acid molecules according toclaim 8, wherein: a. at least one transporter polypeptide is derivedfrom an alloalbumin; b. at least one transporter polypeptide is derivedfrom human serum albumin; c. one of said first transporter polypeptideand said second transporter polypeptide is derived from an alloalbuminand the other is derived from a different alloalbumin, or d. one of saidfirst transporter polypeptide and said second transporter polypeptide isderived from human serum albumin and the other is derived from analloalbumin.
 10. The nucleic acid molecules according to claim 5,wherein the mammalian albumin is human serum albumin.
 11. The nucleicacid molecules according to claim 5, wherein said first transporterpolypeptide has an amino acid sequence comprising SEQ ID NO:2 or ananalog or variant thereof, and wherein said second transporterpolypeptide has an amino acid sequence comprising SEQ ID NO:3 or ananalog or variant thereof.
 12. The nucleic acid molecules according toclaim 11, wherein one nucleic acid has a nucleotide sequence comprisingSEQ ID NO:6 or an analog or variant thereof, and another nucleic acidhas a nucleotide sequence comprising SEQ ID NO:7 or an analog or variantthereof.
 13. The nucleic acid molecules according to claim 5, whereinsaid first transporter polypeptide has an amino acid sequence comprisingSEQ ID NO:8 or an analog or variant thereof, and wherein said secondtransporter polypeptide has an amino acid sequence comprising SEQ IDNO:10 or an analog or variant thereof.
 14. The nucleic acid moleculesaccording to claim 13, wherein one nucleic acid has a nucleotidesequence comprising SEQ ID NO:9 or an analog or variant thereof, andanother nucleic acid has a nucleotide sequence comprising SEQ ID NO:11or an analog or variant thereof.
 15. The nucleic acid moleculesaccording to claim 1, wherein said first transporter polypeptide andsaid second transporter polypeptide are derived from a non-mammalianalbumin.
 16. The nucleic acid molecules according to claim 1, wherein atleast one cargo polypeptide is an antibody, or a fragment or variantthereof.
 17. The one nucleic acid molecules according to claim 1,wherein the at least one cargo polypeptide of said first protein binds atarget antigen, and the at least one cargo polypeptide of said secondprotein comprises a toxin moiety.
 18. The nucleic acid moleculesaccording to claim 1, wherein said at least one cargo polypeptide bindsa target antigen, wherein said target antigen is at least one ofalpha-chain (CD25) of IL-2R, Amyloid beta, anti-EpCAM, anti-CD3, CD16,CD20, CD22, CD23, CD3, CD4, CD52, CD80, CTLA-4, EGFR, EpCAM, F proteinof RSV, G250, glycoprotein IIB/IIIa R, HER2, HSP90, IgE antibody, IL-12,IL-23, IL-1beta, IL-5, IL-6, RANKL, TNF alpha, TNFR, VEGF-A, glucagonreceptor, GLP receptor, and LDL receptor.
 19. The nucleic acid moleculesaccording to claim 1 wherein at least one cargo polypeptide is anenzyme, hormone, therapeutic polypeptide, antigen, chemotoxin, cytokineor a variant or fragment thereof.
 20. The nucleic acid moleculesaccording to claim 1, wherein said first protein and said second proteincomprise the same cargo polypeptide.
 21. The nucleic acid moleculesaccording to claim 1, wherein the cargo polypeptide is attached to thetransporter polypeptide by direct fusion or fusion via a linker.
 22. Thenucleic acid molecules according to claim 21, wherein the linker is aGGSG linker or a (GGSG)4GG linker.
 23. The nucleic acid moleculesaccording to claim 1, wherein the heteromultimer binds to FcRn.
 24. Thenucleic acid molecules according to claim 1, wherein the first proteinand the second protein comprise different cargo polypeptides.
 25. Thenucleic acid molecules according to claim 1, wherein the first proteincomprises at least two different cargo polypeptides, or the secondprotein comprises at least two different cargo polypeptides.
 26. One ormore vectors comprising the nucleic acid molecules according to claim 1.27. A method of expressing a heteromultimer in mammalian or yeast cells,the method comprising: a) transfecting at least one mammalian or yeastcell with the nucleic acid molecules according to claim 1, to produce atleast one transfected mammalian or yeast cell; and b) culturing said atleast one transfected mammalian or yeast cell under conditions suitablefor expressing the heteromultimer.